Benzodiazepinone compounds useful in the treatment of skin conditions

ABSTRACT

The present invention provides a family of benzodiazepinone compounds and pharmaceutical compositions thereof. The present invention also provides methods of treating certain skin conditions, e.g., atopic dermatitis, rosacea, or psoriasis, by administering a benzodiazepinone and methods of reducing the proliferation of keratinocyte cells by exposing such cells to a benzodiazepinone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 13,283,231, filed Oct. 27, 2011, which will issue on Jun. 11,2013 as U.S. Pat. No. 8,461,153, which is a continuation of pending U.S.patent application Ser. No. 12/266,239, filed Nov. 6, 2008, which issuedon May 29, 2012 as U.S. Pat. No. 8,188,072, which claims the benefit ofpriority to expired U.S. Provisional Patent Application No. 60/985,898,filed Nov. 6, 2007, the contents of which are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI047450 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of pharmaceuticalcompounds useful in treating aberrant skin conditions. Morespecifically, the present invention relates to benzodiazepinonecompounds and methods of using the same to treat skin conditionsassociated with epidermal hyperplasia, such as psoriasis.

BACKGROUND OF THE INVENTION

Aberrant skin conditions afflict millions of people worldwide. Patientssuffering from a skin condition often experience pain and/or discomfortdue to the condition. For some patients, the pain or discomfort can bequite severe. A patient's ability to sleep, perform routine tasks,and/or participate in certain sports can be affected by some skinconditions. In addition to physical ailments, skin conditions can have adetrimental affect on a patient's psychological wellbeing. For instance,some skin conditions cause unsightly sores on a patient's skin, causingsome patients to avoid social interaction and/or become emotionallydepressed. Rosacea is one such condition that can have psychologicaleffects because it often causes red discoloration of the face and theappearance of unsightly, acne-like pimples.

One characteristic of numerous skin conditions is epidermal hyperplasia.Epidermal hyperplasia is an abnormal increase in the number of normalcells in normal arrangement in epidermal tissue. Research hasdemonstrated that excessive growth of keratinocyte cells is commonlyassociated with epidermal hyperplasia. It is postulated that epidermalhyperplasia involves a complex multi-cellular inflammatory event.However, topical treatment of all-trans retinoic acid (RA) or itsprecursor, all-trans retinol (ROL), to the skin also results inepidermal hyperplasia. See, e.g., Varani J, et al., (2001) J. Invest.Dermatol, 117:1335-1341. Representative skin conditions associated withepidermal hyperplasia include psoriasis and atopic dermatitis. See,e.g., Krueger G C, et al., (1984) J. Am. Acad. Dermatol. 11: 937-947;Fry L. (1988), Brit. J. Dermatol. 119:445-461

Psoriasis is a chronic, inflammatory, hyperproliferative skin conditionthat affects approximately 2% of the general population. Approximately150,000 new cases of psoriasis and approximately 400 deaths frompsoriasis are reported each year. See Stem, R. S. (1995) Dermatol. Clin.13:717-722. Typical symptoms of psoriasis include skin lesions, redness,inflammation, or patches of skin that become dry, red, covered withsilvery scales, cracked, and/or painful. Additional symptoms includejoint pain or aching, although these symptoms are typically associatedwith psoriatic arthritis. Psoriasis can affect all parts of the skin,but it is more commonly seen on the skin of the trunk, scalp, elbows,knees, or in the fingernails or toenails. The symptoms of psoriasis maybecome worse in response to cuts, burns, insect bites or other skininjuries. The symptoms of psoriasis can also be more severe in patientshaving a deficient immune system, such as patients afflicted with AIDSor receiving cancer chemotherapy.

There are a several of types of psoriasis. The most common type ofpsoriasis is chronic plaque syndrome. This type of psoriasis consists ofperiods of remission and relapse during the course of the condition. Ifleft untreated, plaque psoriasis can evolve into a more severecondition, such as pustular psoriasis or erythrodermic psoriasis. Inpustular psoriasis, the red areas on the skin contain blisters with pus.Erythrodermic psoriasis is characterized by large patches of skin thatare red and scaling. Patients suffering from erythrodermic psoriasisoften complain that the affected patches of skin are itchy and/orpainful. Inverse psoriasis is characterized by smooth, inflamed areas ofskin, typically appearing in skin folds. Guttate psoriasis appears asnumerous, teardrop-shaped spots on the skin and is often associated withstreptococcal throat infection. Nail psoriasis is characterized bychanges in the finger and toe nails. This form of psoriasis ofteninvolves discoloring under the nail or thickening of the skin under thenail.

The current methods for treating psoriasis suffer from a number ofdrawbacks. For example, many of the currently-available, topicalanti-psoriatic agents irritate the skin, cannot be used for extendeddurations, and/or lead to aggressive recurrence of the psoriaticcondition if treatment is terminated abruptly. Anti-inflammatory agents,although capable of alleviating certain symptoms, do not cure theunderlying disease. Another current treatment option, photochemotherapy,can lead to squamous-cell and melanoma skin cancer.

Accordingly, the need exists for new compositions and methods that areeffective in treating skin conditions, such as psoriasis.

SUMMARY OF THE INVENTION

The present invention provides benzodiazepinone compounds, methods fortreating various conditions using benzodiazepinone compounds, andmethods for reducing the proliferation of a keratinocyte cell usingbenzodiazepinone compounds.

In one aspect, the invention provides a compound represented by FormulaI:

-   -   including salts, esters and prodrugs thereof, wherein    -   R¹ is H or C₁₋₆ alkyl;    -   R² is H or C₁₋₆ alkyl;    -   R³ is C₁₋₆ alkyl;    -   R⁴ is hydroxyl or fluoro;    -   the stereochemical configuration at a stereocenter in a compound        represented by formula I is R, S, or a mixture thereof; and    -   provided that said compound is not

The family of compounds embraced by formula I can be present inpharmaceutical compositions comprising a compound described herein and apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition further comprises a steroid, cyclosporine,vitamin D, vitamin D analog, keratolytic agent, topical retinoid,calcineurin inhibitor, or coal tar.

In another aspect, the invention provides a method of treating a skincondition. The method comprises administering a therapeuticallyeffective amount of a compound of formula I, described herein, to asubject in need thereof to ameliorate a symptom of the condition. Avariety of skin conditions can be treated, particularly those associatedwith epidermal hyperplasia. In certain embodiments, the skin conditionis atopic dermatitis, rosacea, or psoriasis.

In another aspect, the invention provides a method of treating epidermalhyperplasia. The method comprises administering a therapeuticallyeffective amount of compound of formula I, described herein, to asubject in need thereof to ameliorate a symptom of the epidermalhyperplasia.

In certain embodiments, the invention provides a combination therapy.For instance, in certain embodiments, one of the aforementioned methodsfurther comprises administering to the subject a therapeutic agentselected from the group consisting of a steroid, cyclosporine, vitaminD, vitamin D analog, keratolytic agent, topical retinoid, calcineurininhibitor, and coal tar. A variety of different steroids, cyclosporines,vitamin D analogs, keratolytic agents, topical retinoids, calcineurininhibitors and coal tars are amenable to the present invention. Incertain embodiments, the steroid is a topical corticosteroid. In certainembodiments, the topical corticosteroid is triamcinolone acetonide orbetamethasone dipropionate; the vitamin D analog is calcipotriene; thekeratolytic agent is anthralin; the topical retinoid is tretinoin ortazarotene; and the calcineurin inhibitor is tacrolimus, pimecrolimus,ascomycin, or ISA247.

In another aspect, the invention provides a method of reducing theproliferation of a keratinocyte cell. The method comprises exposing akeratinocyte cell to a compound of formula I, described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate an understanding of the invention, the following termshave the meanings defined below.

The term “benzodiazepine” refers to a seven-membered non-aromaticheterocyclic ring fused to a phenyl ring wherein the seven-membered ringhas two nitrogen atoms, as part of the heterocyclic ring. The twonitrogen atoms are in 1 and 4 positions, as shown in the generalstructure below.

The benzodiazepine can be substituted with one keto group (typically atthe 2-position), or with two keto groups, one each at the 2- and5-positions. For purposes of the present invention, the benzodiazepinecompounds encompass various substituents at the seven-memberednon-aromatic heterocyclic ring.

The term “alkyl” is art-recognized, and includes saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Representative examples includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl,pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcyclopropylmethyl.

The term “hydroxyl” means —OH. For example, pentane substituted with ahydroxyl group has the formula CH₃(CH₂)₃CH₂OH.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo.

The term “pharmaceutically acceptable carrier” refers to any of thestandard pharmaceutical carriers, such as a phosphate buffered salinesolution, water, emulsions (e.g., such as an oil/water or water/oilemulsions), and various types of wetting agents. The compositions alsocan include stabilizers and preservatives. For examples of carriers,stabilizers and adjuvants. (See e.g., Martin, Remington's PharmaceuticalSciences, 15th Ed., Mack Publ. Co., Easton, Pa. [1975]).

The term “pharmaceutically acceptable salt” refers to anypharmaceutically acceptable salt (e.g., acid or base) of a compound ofthe present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilesometime not in themselves pharmaceutically acceptable, may be employedin the preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts. Examples of bases include alkali metals (e.g., sodium)hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like. Examples of salts include, but are not limited to, acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

It will be noted that the structure of some of the compounds of theinvention includes asymmetric carbon atoms. It is to be understood thatthe isomers arising from such asymmetry (e.g., all enantiomers anddiastereomers) are included within the scope of the invention, unlessindicated otherwise. Such isomers can be obtained in substantially pureform by classical separation techniques and by stereochemicallycontrolled synthesis. Furthermore, the structures and other compoundsand moieties discussed in this application also include all tautomersthereof. Alkenes can include either the E- or Z-geometry, whereappropriate.

The terms ortho, meta and para are art-recognized and refer to 1,2-,1,3- and 1,4-disubstituted benzenes, respectively. For example, thenames 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

The term “hyperplasia” refers to a form of cell proliferation involvingan increase in cell number in a tissue or organ, without significantalteration in structure or function. Pathologically, hyperplasiainvolves the proliferation of cells that is not governed by the usuallimitations of normal cell growth. Hyperplasia may be exhibited inhyperplastic cells or cancer cells, which includes tumor cells,neoplastic cells, malignant cells, and metastatic cells.

The term “epidermal hyperplasia” refers to an abnormal multiplication orincrease in the number of normal cells in normal arrangement inepidermal tissue. Epidermal hyperplasia is a characteristic of numerousskin conditions, including but not limited to, psoriasis.

The term “keratinocyte” refers to a skin cell of the keratinized layerof the epidermis.

The term “fibroblast” refers to mesodermally-derived resident cells ofconnective tissue that secrete fibrillar procollagen, fibronectin andcollegenase.

The term “EC₅₀” is art-recognized and refers to the concentration of acompound at which 50% of its maximal effect is observed.

The term “subject” refers to organisms to be treated by the methods ofthe present invention. Such organisms preferably include mammals (e.g.,murines, simians, equines, bovines, porcines, canines, felines, and thelike), and most preferably includes humans.

The term “effective amount” refers to the amount of a compound (e.g.,benzodiazepinone compound) sufficient to effect beneficial or desiredresults. An effective amount can be administered in one or moreadministrations, applications or dosages and is not limited or intendedto be limited to a particular formulation or administration route.

The term “second agent” refers to a therapeutic agent other than thebenzodiazepinone compounds in accordance with the present invention. Incertain instances, the second agent is an anti-proliferative agent.

The term “co-administration” refers to the administration of at leasttwo agent(s) (e.g., a compound of the present invention) or therapies toa subject. In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. Those ofskill in the art understand that the formulations and/or routes ofadministration of the various agents/therapies used may vary. Theappropriate dosage for co-administration can be readily determined byone skilled in the art. In some embodiments, when agents/therapies areco-administered, the respective agents/therapies are administered atlower dosages than appropriate for their administration alone. Thus,co-administration is especially desirable in embodiments where theco-administration of the agents/therapies lowers the requisite dosage ofa known potentially harmful (e.g., toxic) agent(s).

The term “combination therapy” includes the administration of abenzodiazepinone compound of the invention and at least a second agentas part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). “Combination therapy” may, but generally is not,intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention. “Combination therapy” is intended to embrace administrationof these therapeutic agents in a sequential manner, that is, whereineach therapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical. “Combination therapy” alsocan embrace the administration of the therapeutic agents as describedabove in further combination with other biologically active ingredientsand non-drug therapies (e.g., surgery or radiation treatment.) Where thecombination therapy further comprises a non-drug treatment, the non-drugtreatment may be conducted at any suitable time so long as a beneficialeffect from the co-action of the combination of the therapeutic agentsand non-drug treatment is achieved. For example, in appropriate cases,the beneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

The present invention provides benzodiazepinone compounds and methods ofusing such compounds as therapeutic agents to treat a number ofdifferent conditions. Exemplary compositions and methods of the presentinvention are described in more detail in the following sections: I.Benzodiazepinone Compounds; II. Therapeutic Applications ofBenzodiazepinone Compounds; and III. Pharmaceutical Compositions,Formulations, and Exemplary Administration Routes and DosingConsiderations.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as“Comprehensive Organic Synthesis” (B. M. Trost & I. Fleming, eds.,1991-1992); “Molecular cloning: a laboratory manual” Second Edition(Sambrook et al., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed.,1984); “Animal cell culture” (R. I. Freshney, ed., 1987); the series“Methods in enzymology” (Academic Press, Inc.); “Handbook ofexperimental immunology” (D. M. Weir & C. C. Blackwell, eds.); “Genetransfer vectors for mammalian cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current protocols in molecular biology” (F. M. Ausubel et al.,eds., 1987, and periodic updates); “PCR: the polymerase chain reaction”(Mullis et al., eds., 1994); and “Current protocols in immunology” (J.E. Coligan et al., eds., 1991), each of which is herein incorporated byreference in its entirety.

I. Benzodiazepinone Compounds

In one aspect, the invention provides a compound represented by FormulaI:

-   -   including salts, esters and prodrugs thereof, wherein    -   R¹ is H or C₁₋₆ alkyl;    -   R² is H or C₁₋₆ alkyl;    -   R³ is C₁₋₆ alkyl;    -   R⁴ is hydroxyl or fluoro;    -   the stereochemical configuration at a stereocenter in a compound        represented by formula I is R, S, or a mixture thereof; and    -   provided that said compound is not

In certain embodiments, R₄ is fluoro. In certain other embodiments, thecompound has the structure:

In certain other embodiments, R₄ is hydroxyl. In certain otherembodiments, the compound has the structure:

In certain other embodiments, the compound has the structure:

In certain other embodiments, R² and R³ are C₁₋₃ alkyl. In certain otherembodiments, R² and R³ are methyl. In certain other embodiments, R² andR³ are ethyl. In certain other embodiments, R² and R³ are isopropyl. Incertain other embodiments, R¹ is methyl. In certain other embodiments,R² is H, and R³ is isopropyl. In certain other embodiments, R¹ is H ormethyl; and R² and R³ are C₁₋₃ alkyl. In certain other embodiments, thecompound is:

In certain other embodiments, the compound is:

In certain embodiments, the compounds are as described in the followingtables.

TABLE 1

No. R¹ R² R³ 1 H 2-CH₃ H 2 H 3-CH₃ H 3 H 4-CH₃ H 4 H 2-CH₃ 3-CH₃ 5 H2-CH₃ 4-CH₃ 6 H 2-CH₃ 5-CH₃ 7 H 2-CH₃ 6-CH₃ 8 H 3-CH₃ 4-CH₃ 9 H 3-CH₃5-CH₃ 10 H 2-CH₂CH₃ H 11 H 3-CH₂CH₃ H 12 H 4-CH₂CH₃ H 13 H 2-CH₂CH₃3-CH₂CH₃ 14 H 2-CH₂CH₃ 4-CH₂CH₃ 15 H 2-CH₂CH₃ 5-CH₂CH₃ 16 H 2-CH₂CH₃6-CH₂CH₃ 17 H 3-CH₂CH₃ 4-CH₂CH₃ 18 H 3-CH₂CH₃ 5-CH₂CH₃ 19 H 2-CH(CH₃)₂ H20 H 3-CH(CH₃)₂ H 21 H 4-CH(CH₃)₂ H 22 H 2-CH(CH₃)₂ 3-CH(CH₃)₂ 23 H2-CH(CH₃)₂ 4-CH(CH₃)₂ 24 H 2-CH(CH₃)₂ 5-CH(CH₃)₂ 25 H 2-CH(CH₃)₂6-CH(CH₃)₂ 26 H 3-CH(CH₃)₂ 4-CH(CH₃)₂ 27 H 3-CH(CH₃)₂ 5-CH(CH₃)₂ 28 CH₃2-CH₃ H 29 CH₃ 3-CH₃ H 30 CH₃ 4-CH₃ H 31 CH₃ 2-CH₃ 3-CH₃ 32 CH₃ 2-CH₃4-CH₃ 33 CH₃ 2-CH₃ 6-CH₃ 34 CH₃ 3-CH₃ 5-CH₃ 35 CH₃ 2-CH₂CH₃ H 36 CH₃3-CH₂CH₃ H 37 CH₃ 4-CH₂CH₃ H 38 CH₃ 2-CH₂CH₃ 3-CH₂CH₃ 39 CH₃ 2-CH₂CH₃4-CH₂CH₃ 40 CH₃ 2-CH₂CH₃ 5-CH₂CH₃ 41 CH₃ 2-CH₂CH₃ 6-CH₂CH₃ 42 CH₃3-CH₂CH₃ 4-CH₂CH₃ 43 CH₃ 3-CH₂CH₃ 5-CH₂CH₃ 44 CH₃ 2-CH(CH₃)₂ H 45 CH₃3-CH(CH₃)₂ H 46 CH₃ 4-CH(CH₃)₂ H 47 CH₃ 2-CH(CH₃)₂ 3-CH(CH₃)₂ 48 CH₃2-CH(CH₃)₂ 4-CH(CH₃)₂ 49 CH₃ 2-CH(CH₃)₂ 5-CH(CH₃)₂ 50 CH₃ 2-CH(CH₃)₂6-CH(CH₃)₂ 51 CH₃ 3-CH(CH₃)₂ 4-CH(CH₃)₂ 52 CH₃ 3-CH(CH₃)₂ 5-CH(CH₃)₂

TABLE 2

No. R¹ R² R³ 1 H 2-CH₃ H 2 H 3-CH₃ H 3 H 4-CH₃ H 4 H 2-CH₃ 3-CH₃ 5 H2-CH₃ 4-CH₃ 6 H 2-CH₃ 5-CH₃ 7 H 2-CH₃ 6-CH₃ 8 H 3-CH₃ 4-CH₃ 9 H 3-CH₃5-CH₃ 10 H 2-CH₂CH₃ H 11 H 3-CH₂CH₃ H 12 H 4-CH₂CH₃ H 13 H 2-CH₂CH₃3-CH₂CH₃ 14 H 2-CH₂CH₃ 4-CH₂CH₃ 15 H 2-CH₂CH₃ 5-CH₂CH₃ 16 H 2-CH₂CH₃6-CH₂CH₃ 17 H 3-CH₂CH₃ 4-CH₂CH₃ 18 H 3-CH₂CH₃ 5-CH₂CH₃ 19 H 2-CH(CH₃)₂ H20 H 3-CH(CH₃)₂ H 21 H 4-CH(CH₃)₂ H 22 H 2-CH(CH₃)₂ 3-CH(CH₃)₂ 23 H2-CH(CH₃)₂ 4-CH(CH₃)₂ 24 H 2-CH(CH₃)₂ 5-CH(CH₃)₂ 25 H 2-CH(CH₃)₂6-CH(CH₃)₂ 26 H 3-CH(CH₃)₂ 4-CH(CH₃)₂ 27 H 3-CH(CH₃)₂ 5-CH(CH₃)₂ 28 CH₃2-CH₃ H 29 CH₃ 3-CH₃ H 30 CH₃ 4-CH₃ H 31 CH₃ 2-CH₃ 3-CH₃ 32 CH₃ 2-CH₃4-CH₃ 33 CH₃ 2-CH₃ 5-CH₃ 34 CH₃ 2-CH₃ 6-CH₃ 35 CH₃ 3-CH₃ 4-CH₃ 36 CH₃3-CH₃ 5-CH₃ 37 CH₃ 2-CH₂CH₃ H 38 CH₃ 3-CH₂CH₃ H 39 CH₃ 4-CH₂CH₃ H 40 CH₃2-CH₂CH₃ 3-CH₂CH₃ 41 CH₃ 2-CH₂CH₃ 4-CH₂CH₃ 42 CH₃ 2-CH₂CH₃ 5-CH₂CH₃ 43CH₃ 2-CH₂CH₃ 6-CH₂CH₃ 44 CH₃ 3-CH₂CH₃ 4-CH₂CH₃ 45 CH₃ 3-CH₂CH₃ 5-CH₂CH₃46 CH₃ 2-CH(CH₃)₂ H 47 CH₃ 3-CH(CH₃)₂ H 48 CH₃ 4-CH(CH₃)₂ H 49 CH₃2-CH(CH₃)₂ 3-CH(CH₃)₂ 50 CH₃ 2-CH(CH₃)₂ 4-CH(CH₃)₂ 51 CH₃ 2-CH(CH₃)₂5-CH(CH₃)₂ 52 CH₃ 2-CH(CH₃)₂ 6-CH(CH₃)₂ 53 CH₃ 3-CH(CH₃)₂ 4-CH(CH₃)₂ 54CH₃ 3-CH(CH₃)₂ 5-CH(CH₃)₂

The foregoing compounds can be present in pharmaceutical compositionscomprising a compound described herein and a pharmaceutically acceptablecarrier. In certain embodiments, the pharmaceutical composition furthercomprises a second therapeutic agent. In certain embodiments, the secondtherapeutic agent is a steroid, cyclosporine, vitamin D, vitamin Danalog, keratolytic agent, topical retinoid, calcineurin inhibitor, orcoal tar. A variety of different steroids, cyclosporines, vitamin Danalogs, keratolytic agents, topical retinoids, calcineurin inhibitors,and coal tars are amenable to the present invention. In certainembodiments, the steroid is a topical steroid. Topical steroids canreduce plaque formation and have anti-inflammatory affects. Topicalsteroids may also modify the body's immune response to diverse stimuli.The topical steroid may, in certain embodiments, be a topicalcorticosteroid, such as triamcinolone acetonide (Artistocort, Kenalog)(0.1% cream) or betamethasone diproprionate (Diprolene, Diprosone)(0.05% cream). In certain embodiments, the second therapeutic agent is acyclosporine, such as Cyclosporine A or a derivative of cyclosporine A.In certain embodiments, the second therapeutic agent is vitamin D or avitamin D analog. Vitamin D analogs are sometimes used in patients withlesions resistant to older therapeutics or with lesions on the face orexposed areas where thinning of the skin would pose cosmetic problems.In certain embodiments, the vitamin D analog is calcipotriene. Incertain embodiments, the second therapeutic agent is a keratolyticagent. Keratolytic agents can be used to remove scale, smooth the skin,and to treat hyperkeratosis in a subject. In certain embodiments, thekeratolytic agent is anthralin, particularly anthralin 0.1-1%(Drithocreme, Anthra-Derm).

In certain embodiments, the second therapeutic agent is a retinoid, suchas a topical retinoid. Topical retinoids can decrease the cohesivenessof follicular epithelial cells and stimulate mitotic, resulting in anincrease in turnover of follicular epithelial cells. In certainembodiments, the topical retinoid is tretinoin or tazarotene. In certainembodiments, the topical retinoid is tretinoin in the formulationmarketed as Retin-A or Avita. In certain embodiments, the topicalretinoid is tazarotene in the formulation marketed as Tazorac.

In certain embodiments, the second therapeutic agent is a calcineurininhibitor, such as tacrolimus, pimecrolimus, ascomycin, or ISA247.Tacrolimus, also known as FK506, is described in U.S. Pat. Nos.4,894,366; 4,916,138; and 4,929,611; each of which is herebyincorporated by reference. Pimecrolimus is a macrolactam that has beenreported to inhibit production of pro-inflammatory cytokines by T cellsand mast cells. Pimecrolimus is described in U.S. Pat. No. 5,912,238,which is hereby incorporated by reference. Ascomycin is an ethyl analogof tacrolimus described by C. E. Griffiths in Br. J. Dermatol. 2001.April; 144(4):679-81. ISA247 is a cyclosporin derivative described inU.S. Pat. Nos. 6,605,593 and 6,613,739; each of which is herebyincorporated by reference.

In certain embodiments, the second therapeutic agent is a coal tar. Coaltar is an inexpensive treatment available over the counter in shampoosor lotions, and it is particularly useful in hair-bearing areas. Oneexample of a coal tar is coal tar (DHS Tar, Doctar, TheraplexT)-antipruitic. In certain embodiments, the coal tar is present at aconcentration of 2-10 percent by weight.

The benzodiazepinone compounds described above can be prepared based onthe procedures depicted in Scheme 1.

N-alkylation of isatoic anhydride A can be carried out by treatingcompound A with sodium hydride and an alkyl or benzyl halide. Reactionof compound A with a benzyl halide, such as a p-methoxybenzyl halide,can be performed to install a protecting group, while reaction ofcompound A with various alkyl halides, e.g., methyl iodide or ethyliodide, can be performed to install alkyl substitution on theN1-position of the benzodiazepine ring. Isatoic anhydride B can beconverted to benzodiazepinone C upon reaction with glycine. See IndianJ. Chem. Sect. B. 1985, 24, 905-907. This procedure providedbenzodiazepinones C1 and C2 in good yield, which were subsequentlytreated with POCl₃ to provide imidoyl chlorides D1 or D2.

The “southern” aryl ring (substituent Ar) can be installed by Suzukicoupling of an aryl boronic acid, in accordance with proceduresdescribed by Nadin and co-workers. See J. Org. Chem. 2003, 68,2844-2852. In particular, Suzuki coupling of imidoyl chlorides D1 and D2with (4-methoxyphenyl)boronic acid afforded 5-arylbenzodiazepinones E1and E2 in bulk quantities.

The “eastern” aryl ring (substituent Ar₂) can be installed by alkylationat the C3-position of the benzodiazepinone ring. Deprotonation at C-3using a strong base, such as potassium tert-butoxide, following by theaddition of a substituted benzyl halide provided benzodiazepinone G. Thebenzyl halides for this reaction can be obtained commercially orprepared from the corresponding benzyl alcohol using known procedures,such as treating a benzyl alcohol with thionyl chloride. A variety ofbenzyl alcohols are commercially available. In addition, a variety ofbenzyl alcohols can be prepared using any one of the following methods:i) reduction of a commercially available carboxylic acid (e.g.,reduction using lithium aluminum hydride); ii) conversion of adibromo-benzyl alcohol to a dialkyl-benzyl alcohol using, for example, adialkylzinc reagent in the presence of a palladium catalyst, such asPdCl₂(dppf); iii) conversion of a dibromobenzyl acetate to a dialkylbenzyl acetate followed by hydrolysis; iv) formylation of theappropriate aromatic compound followed by reduction; or v) conversion ofa reactive chlorobenzoate ester to the respective alkyl benzoate esterusing, for example, a Grignard reagent in the presence of an ironcatalyst, such as Fe(acac)₃, followed by reduction.

Substituents on the “eastern” aromatic ring can be installed followingC3-alkylation of the aromatic ring. For example, C3-alkylation with3-bromobenzyl bromide, followed by Pd-catalyzed attachment of an alkylgroup to the aromatic ring.

As illustrated in Scheme 1 above, benzodiazepinone G can also beprepared using a synthetic strategy involving C3-alkylation of imidoylchloride D followed by a palladium-coupling reaction to install the“southern” aromatic ring. This synthetic strategy should be amenable towide a variety of substrates, although aqueous work-ups of theC3-alkylation reaction should be performed quickly at low temperature tominimize any hydrolysis of the imidoyl chloride group. The arylboronates used in this palladium-coupling reaction can be obtained fromcommercial sources or they can be easily prepared. For example, an arylboronate can be prepared by treating an aryl bromide withbis(pinacolato)diboron in the presence of a palladium catalyst.

In situations where protecting groups are used during the synthesis,protecting groups on compound G can be removed using standard proceduresknown in the art. For example, the methoxy protecting group in Ar₁ canbe removed using AlCl₃ to afford the p-hydroxyphenyl group. Similarly,N-deprotection of a p-methoxybenzyl group can be performed using cerium(IV) ammonium nitrate, according to literature procedures.

II. Therapeutic Applications of Benzodiazepinone Compounds

It is contemplated that the benzodiazepinone compounds of Formula Iprovide therapeutic benefits to patients suffering from various skinconditions. In one aspect, the invention provides a method of treating askin condition, comprising administering a therapeutically effectiveamount of a compound of formula I to a subject in need thereof toameliorate a symptom of the condition:

-   -   including salts, esters and prodrugs thereof, wherein    -   R¹ is H or C₁₋₆ alkyl;    -   R² is H or C₁₋₆ alkyl;    -   R³ is C₁₋₆ alkyl;    -   R⁴ is hydroxyl or fluoro;    -   the stereochemical configuration at a stereocenter in a compound        represented by formula I is R, S, or a mixture thereof; and    -   provided that said compound is not

In certain embodiments, R₄ is fluoro. In certain other embodiments, thecompound has the structure:

In certain other embodiments, R₄ is hydroxyl. In certain otherembodiments, the compound has the structure:

In certain other embodiments, the compound has the structure:

In certain other embodiments, R² and R³ are C₁₋₃ alkyl. In certain otherembodiments, R² and R³ are methyl. In certain other embodiments, R² andR³ are ethyl. In certain other embodiments, R² and R³ are isopropyl. Incertain other embodiments, R¹ is methyl. In certain other embodiments,R² is H, and R³ is isopropyl. In certain other embodiments, R¹ is H ormethyl; and R² and R³ are C₁₋₃ alkyl. In certain other embodiments, thecompound is:

In certain other embodiments, the compound is:

In certain other embodiments, the compound is one of the compoundslisted in Tables 1 or 2. In certain other embodiments, the skincondition is associated with epidermal hyperplasia. In certain otherembodiments, the skin condition is atopic dermatitis, rosacea orpsoriasis. In certain other embodiments, the skin condition ispsoriasis. There are a variety of forms of psoriasis, including plaquepsoriasis, guttate psoriasis, nail psoriasis, inverse psoriasis, andscalp psoriasis. It is contemplated that one or more of these forms ofpsoriasis can be treated by administering a benzodiazepinone describedherein.

In another aspect, the present invention provides a method of treatingepidermal hyperplasia, comprising administering a therapeuticallyeffective amount of a compound of formula I, described herein, to asubject in need thereof to ameliorate a symptom of the epidermalhyperplasia. In certain embodiments, the compound has the structure:

wherein R¹ is H or C₁₋₆ alkyl; R² is H or C₁₋₆ alkyl; R³ is C₁₋₆ alkyl;and the stereochemical configuration at a stereocenter in the compoundis R, S, or a mixture thereof.

In certain other embodiments, the compound has the structure:

wherein R¹ is H or C₁₋₆ alkyl; R² is H or C₁₋₆ alkyl; R³ is C₁₋₆ alkyl;and the stereochemical configuration at a stereocenter in the compoundis R, S, or a mixture thereof.

In certain other embodiments, R² and R³ are C₁₋₃ alkyl. In certain otherembodiments, R² and R³ are methyl. In certain other embodiments, R² andR³ are ethyl. In certain other embodiments, R² and R³ are isopropyl. Incertain other embodiments, R¹ is methyl. In certain other embodiments,R² is H, and R³ is isopropyl. In certain other embodiments, R¹ is H ormethyl; and R² and R³ are C₁₋₃ alkyl. In certain other embodiments, thecompound is:

In certain other embodiments, the compound is:

In certain other embodiments, the compound is one of the compoundslisted in Tables 1 or 2.

In certain embodiments, the subject is a human. In certain otherembodiments, the method further comprises administering to the subject atherapeutic agent selected from the group consisting of a steroid,cyclosporine, vitamin D, vitamin D analog, keratolytic agent, topicalretinoid, calcineurin inhibitor, and coal tar. In certain embodiments,the steroid is a topical corticosteroid. In certain other embodiments,the topical corticosteroid is triamcinolone acetonide or betamethasonedipropionate; the vitamin D analog is calcipotriene; the keratolyticagent is anthralin; the topical retinoid is tretinoin or tazarotene; andthe calcineurin inhibitor is tacrolimus, pimecrolimus, ascomycin, orISA247. In certain other embodiments, the therapeutic agent is asteroid.

In another aspect, the present invention provides a method of reducingthe proliferation of a keratinocyte cell, comprising exposing said cellto a compound of formula I, described herein. In certain embodiments,the compound has the structure:

wherein R¹ is H or C₁₋₆ alkyl; R² is H or C₁₋₆ alkyl; R³ is C₁₋₆ alkyl;and the stereochemical configuration at a stereocenter in the compoundis R, S, or a mixture thereof.

In certain other embodiments, the compound has the structure:

wherein R¹ is H or C₁₋₆ alkyl; R² is H or C₁₋₆ alkyl; R³ is C₁₋₆ alkyl;and the stereochemical configuration at a stereocenter in the compoundis R, S, or a mixture thereof.

In certain other embodiments, R² and R³ are C₁₋₃ alkyl. In certain otherembodiments, R² and R³ are methyl. In certain other embodiments, R² andR³ are ethyl. In certain other embodiments, R² and R³ are isopropyl. Incertain other embodiments, R¹ is methyl. In certain other embodiments,R² is H, and R³ is isopropyl. In certain other embodiments, R¹ is H ormethyl; and R² and R³ are C₁₋₃ alkyl. In certain other embodiments, thecompound is:

In certain other embodiments, the compound is:

In certain other embodiments, the compound is one of the compoundslisted in Tables 1 or 2.

In certain other embodiments, a method described herein furthercomprises, exposing said subject to ultraviolet radiation. Phototherapyhas been shown to help treat psoriasis. There are two main forms ofphototherapy, UVB and PUVA phototherapy. UVB, or Ultraviolet B,phototherapy uses light having a wavelength in the range of 290-320 nm.Such phototherapy is often combined with one or more topical treatmentsincluding: i) topically applying coal tar, followed by using UVB; ii)using a coal tar bath, followed by UVB, and then topically applyinganthralin; or iii) using UVB in combination with topically applyingcorticosteroids, calcipotriene, tazarotene, or simply bland emollients.PUVA uses the photosensitizing drug methoxsalen (8-methoxypsoralens) inconjunction with UVA light (wavelengths in the 320-400 nm range). PUVAis thought to interfere with DNA synthesis (methoxsalen binds covalentlyto pyrimidine bases in DNA), decrease cellular proliferation, and induceapoptosis of cutaneous lymphocytes leading to localizedimmunosuppression.

III. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

The compounds of the present invention are useful in the preparation ofmedicaments to treat or study a variety of skin conditions. In certainembodiments, the skin condition is associated with epidermalhyperplasia.

In addition, the compounds are also useful for preparing medicaments fortreating or studying other skin conditions wherein the effectiveness ofthe compounds are known or predicted. The methods and techniques forpreparing medicaments of a compound of the present invention arewell-known in the art. Exemplary pharmaceutical formulations and routesof delivery are described below.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent, as discussed above, together with asolid support or alternatively, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticagents. Each carrier should be “acceptable” in the sense that it iscompatible with the other ingredients of the formulation and notinjurious to the subject.

Contemplated formulations include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In some embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets are made by molding in a suitablemachine a mixture of the powdered compound (e.g., active ingredient)moistened with an inert liquid diluent. Tablets may optionally be coatedor scored and may be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternative embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In some embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in a known manner. This phasetypically comprises a lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprise a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. In some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, and include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administertherapeutic agents (e.g., exemplary compounds as described above) of thepresent invention, e.g., encapsulation in liposomes, microparticles,microcapsules, receptor-mediated endocytosis, and the like. Methods ofdelivery include, but are not limited to, intra-arterial,intra-muscular, intravenous, intranasal, and oral routes. In specificembodiments, it may be desirable to administer the pharmaceuticalcompositions of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, injection, or by means of a catheter.

The agents identified can be administered to subjects or individualssusceptible to or at risk of developing pathological growth of targetcells and correlated conditions. When the agent is administered to asubject such as a mouse, a rat or a human patient, the agent can beadded to a pharmaceutically acceptable carrier and systemically ortopically administered to the subject. To identify patients that can bebeneficially treated, a tissue sample is removed from the patient andthe cells are assayed for sensitivity to the agent.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor non-aqueous diluents, syrups, granulates or powders. In addition toan agent of the present invention, the pharmaceutical compositions canalso contain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or by oral administration, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

D. Exemplary Co-administration Routes and Dosing Considerations

As described above, the invention includes methods involvingco-administration of the compounds described herein with one or moreadditional active agents. Indeed, it is a further aspect of thisinvention to provide methods for enhancing prior art therapies and/orpharmaceutical compositions by co-administering a compound of thisinvention. In co-administration procedures, the agents may beadministered concurrently or sequentially. In one embodiment, thecompounds described herein are administered prior to the other activeagent(s). The pharmaceutical formulations and modes of administrationmay be any of those described above. The determination of appropriatetype and dosage of radiation treatment is also within the skill in theart or can be determined with relative ease.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases wheredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.

EXAMPLES

The invention, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Representative Procedures for N-Alkylation of IsatoicAnhydrides

6-Chloro-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (B1)

In a 3 L, 3-neck RBF equipped with mechanical stir, addition funnel,thermocouple and N₂ inlet, NaH (30.4 g) was suspended in anhydroustetrahydrofuran (THF, 400 mL). While stirring at room temperature, asuspension of 5-chloroisotonic anhydride in THF (400 mL) was added inportion-wise manner over 45 min. The reaction mixture was stirred for 50min (reaction temperature went up from 18 to 28° C.). To this was addedCH₃I (285 g, 125 mL) over 15 min. The mixture was then stirred at 42° C.for 16 h. Because TLC showed that some unreacted starting material wasstill present in the reaction mixture, an additional 30 mL of CH₃I wasadded and the reaction mixture stirred at 42° C. for an additional 3 h.Reaction mixture was cooled (RT) and quenched by the slow (40 min)addition of AcOH (55 mL). Reaction mixture was concentrated to give 275g thick syrupy product, which was used without any further purification.¹H NMR (300 MHz, CDCl₃) δ 3.35 (s, 3H), 7.54 (d, 1H), 7.85 (d, 1H), 7.90(s, 1H).

6-Chloro-1-(4-methoxybenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione (B2)

In a 3 L, 3-neck RBF equipped with mechanical stir, thermocouple and N₂inlet, 90 g (0.455 mol) of 5-chloroisotonic anhydride was suspended inanhydrous THF (0.9 L). Under N₂, 4-methoxybenzylchloride (75 g, 0.48mol) was added followed by the addition of tetrabutylammonium iodide (84g, 0.23 mol). The reaction mixture was stirred for 5 min at roomtemperature and then 20 g (0.5 mol) of NaH was added portion-wise over20 min (reaction temperature increased to 29° C. due to an exotherm andtherefore reaction mixture was placed into a water bath to keep thetemperature below 30° C.). Reaction was stirred for 16 h (RT). The nextday, HPLC showed about 26% unreacted 5-chloroisotonic anhydride.Additional NaH (1 g) was added and the reaction mixture was heated to32° C. and stirred for another 5 h. NMR showed that all of the startingmaterial had been consumed. Reaction was quenched by adding 10 g ofglacial acetic acid slowly, followed by stirring for 30 min. Reactionmixture was filtered through celite, and the filter cake was washed withTHF. Filtrate was concentrated to give 280 g of crude product(yellow-brown solid), which was used with no further purification. ¹HNMR (300 MHz, DMSO-d₆) δ 3.8 (s, 3H), 5.25 (s, 2H), 6.8 (d, 2H), 7.2 (m,3H), 7.75 (d, 1H), 7.9 (d, 1H).

Example 2 Representative Procedures for Conversion of an IsatoicAnhydride to a Benzodiazepine-dione

7-Chloro-1-methyl-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione (C1)

In a 2 L RBF equipped with mechanical stir, condenser and N₂ inlet,glycine (38 g, 0.506 mol) was added to crude B1 (107 g, 0.506 mol)followed by the addition of AcOH (500 mL). Reaction flask was heated ina 130° C. oil bath for 7 h. Solvent was evaporated under suction withheating (50-60° C.). To the thick syrupy crude product was added 1 L ofEtOAc followed by the slow addition of aqueous NaHCO₃ (saturated) toadjust the pH to −7. Then 10 mL of 2 M NaOH was added to adjust the pHto ˜9-10. The mixture gave a solid along with organic and aqueouslayers. The solid was filtered to give product containing some impurity.The solid was partitioned between 400 mL dichloromethane (DCM) and 200mL NaHCO₃, and the resultant slurry was stirred for 20 min, thenfiltered to remove an insoluble impurity. The DCM layer was separatedand washed with 3% NaHCO₃ and then brine (200 mL). The DCM layer wasdried (MgSO₄), filtered and concentrated to give 50 g of pure product.EtOAc layer was concentrated to give 67 g of solid product with someimpurity. The aqueous layer was extracted with EtOAc (2×400 mL).Combined organics were dried over Na₂SO₄, filtered and concentrated togive an additional 6.7 g of crude product. Total of 123.4 g of productwas obtained, 50 g of which was very clean (yield ˜quantitative). ¹H NMR(300 MHz, CDCl₃) δ 3.2 (s, 3H), 3.5 (m, 1H), 3.8 (m, 1H), 7.35 (d, 1H),7.6 (m, 2H), 8.8 (t, 1H).

7-Chloro-1-(4-methoxybenzyl)-3,4-dihydro-1H-benzo[e][1,4]diazepine-2,5-dione(C2)

In a 2 L RBF equipped with mechanical stir, condenser and N₂ inlet,glycine (34 g, 0.45 mol) was added to B2 (280 g) followed by theaddition of AcOH (500 mL). Reaction flask was heated in a 130° C. oilbath for 8 h. Solvent was removed on the rotary evaporator at 50-60° C.To the thick syrupy crude product was added heptane (1 L) and H₂O (1 L)followed by the addition of NaHCO₃ to adjust the pH to ˜8-9. The mixturegave a solid along with organic and aqueous layers. The organic andaqueous layers were decanted and the solid was slurried with 500 mL of5% NaHCO₃ solution. NaHCO₃ layer was decant and sticky solid wassuspended in 700 mL ethylacetate (EtOAc) and 300 mL of dichloromethane(DCM). The mixture was stirred for 20 min, filtered and the filter cakewas washed with 1 L of DCM. The filtrate was concentrated and residuewas pass through 330 g silica gel plug using 25/75 to 75/25EtOAc/heptane (total of 8 L). Clean fractions were combined to give 58 gof pure product. An additional 13 g of −70% pure product was obtainedfrom less pure fractions. Yield was 47% over two steps. ¹H NMR (300 MHz,DMSO-d₆) δ 3.45 (m, 1H), 3.6 (s, 3H), 3.8 (m, 1H), 4.8 (d, 1H), 5.3 (d,1H), 6.8 (d, 2H), 7.1 (d, 2H), 7.7-7.5 (m, 3H), 8.9 (t, 1H).

Example 3 Representative Procedures for Synthesis of an Imidoyl Chloride

(E)-5,7-Dichloro-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one (D1)

In a 1 L 2-neck RBF equipped with mechanical stir, condenser and N₂inlet, C1 (42.5 g, 0.189 mol) was suspended into 400 mL of toluene. Tothis was added N,N-dimethylanaline (45.5 g. 0.375 mol) followed by theaddition of POCl₃ (29 g, 0.189 mol) and the reaction mixture stirred for3 minutes at room temperature (RT). Reaction flask was placed in a 90°C. oil bath and the reaction mixture stirred/heated for 7 h and then atRT for 9 h. The reaction was quenched by adding 500 mL of ice water andstirred for 15 min. Organic layer was separated and quickly washed withcold 0.5 M HCl (300 mL), cold water (300 mL), and then cold saturatedNaHCO₃ (300 mL). Organic layer was dried (MgSO₄), filtered andconcentrated on a rotary evaporator to give 40 g of yellow solid. Yield87.5%. ¹H NMR (300 MHz, DMSO-d₆) δ 3.25 (s, 3H), 3.8-3.9 (s, 1H, br),4.3-4.4 (s, 1H, br), 7.4 (d, 1H), 7.7-7.8 (m, 2H).

(E)-5,7-Dichloro-1-(4-methoxybenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

In a 1 L 3-neck RBF equipped with magnetic stir bar, condenser and N₂inlet, C2 (45 g, 0.136 mol) was suspended in 400 mL of toluene. To thiswas added N,N-dimethylanaline (33 g, 0.272 mol) followed by the additionof POCl₃ (23 g) and the reaction stirred for 3 min (RT). Reaction flaskwas placed into a 90° C. oil bath and the reaction mixture was heatedfor 5 h and then cooled. The reaction was quenched by adding 450 mL ofice water and stirred for 15 min. Organic layer was separated andquickly washed with cold water (2×250 mL) and brine (300 mL). Organiclayer was dried over MgSO₄, filtered and concentrated on a rotaryevaporator to give 57 g of black crude product. Crude product was usedfor next step with no further purification. Yield 87.5%.

Example 4 Representative Procedures for the Preparation of BenzylHalides

Benzyl halides can prepared from the corresponding benzyl alcohol usingknown procedures, such as by treating a benzyl alcohol with thionylchloride. A variety of benzyl alcohols are commercially available. Inaddition, a variety of benzyl alcohols can be prepared using thefollowing methods: i) reduction of a commercially available carboxylicacid (e.g., reduction using lithium aluminum hydride); ii) conversion ofa dibromo-benzyl alcohol to a dialkyl-benzyl alcohol using, for example,a dialkylzinc reagent in the presence of a palladium catalyst, such asPdCl₂(dppf); iii) conversion of a dibromobenzyl acetate to a dialkylbenzyl acetate followed by hydrolysis; iv) formylation of theappropriate aromatic followed by reduction; or v) conversion of areactive chlorobenzoate ester to the respective alkyl benzoate esterusing, for example, a Grignard reagent in the presence of an ironcatalyst, such as Fe(acac)₃, followed by reduction.

Part I: Representative Procedures for the Preparation of a SubstitutedBenzyl Alcohol From Dibromotoluene.

1,3-Dibromo-2-(bromomethyl)benzene

A mixture of 2,6-dibromotoluene (22.9 g, 92 mmol), N-bromosuccinimide(NBS) (15 g, 84 mmol), CCl₄ (250 mL) and benzoyl peroxide (0.03 eq) wasstirred at 85° C. (hot oil bath temperature) for 16 h, cooled to RT,filtered, washed with aq. NaHSO₃, dried (Na₂SO₄), filtered, andevaporated to give 29.5 g (yield of 98%) of title product as a whitesolid. This solid contained 10% unreacted starting material but wassuccessfully used without further purification. ¹H NMR (300 MHz, CDCl₃)δ 4.81 (s, 2H), 7.01 (t, 1H), 7.53 (d, 2H).

2,6-Dibromobenzyl acetate

A mixture of 1,3-dibromo-2-(bromomethyl)benzene (27.6 g, 84 mmol), NaOAc(35.5 g, 5 eq.) and dimethylformamide (DMF) (150 mL) was stirred at 100°C. (hot oil bath temperature) for 1.75 h, allowed to cool, and thenpartitioned between heptane (500 mL) and water (200 mL). After removingthe organic layer, the aqueous layer was extracted with heptane (200mL). The combined organics were washed with H₂O (2×300 mL), dried(Na₂SO₄), filtered, and evaporated to give 24.57 g (yield of 95%) oftitle product as a colorless oil. This oil contained 13% unreactedstarting material but was successfully used without furtherpurification. ¹H NMR (300 MHz, CDCl₃) δ 2.10 (s, 3H), 5.41 (s, 2H), 7.08(t, 1H), 7.58 (d, 2H).

2,6-Diethylbenzyl acetate

To a cooled (dry ice-acetone bath) mixture of 2,6-dibromo-benzyl acetate(5.05 g, 16.4 mmol) and PdCl₂(dppf) (0.08 eq) in dry THF (50 mL) wasadded 1.1 M Et₂Zn (60 mL, 66 mmol, 4 eq). The resulting mixture wasallowed to warm to RT, stirred at 45° C. (programmed block temperature,˜40 h), and added to a stirred mixture of dilute HCl and heptane/EtOAc.The organic layer was dried (Na₂SO₄), filtered, and evaporated.Chromatography (2%-10% EtOAc/heptane stepwise gradient) gave 2.12 g(yield of 63%) of the title product, along with 0.20 g of2,6-diethylbenzyl alcohol. ¹H NMR (300 MHz, CDCl₃) δ 1.21 (t, 6H), 2.07(s, 3H), 2.70 (q, 4H), 5.19 (s, 2H), 7.09 (d, 2H), 7.22 (dd, 1H). Thefollowing compound was prepared by making appropriate substitutions tothe above procedure:

2,6-dimethylbenzyl acetate

¹H NMR (300 MHz, CDCl₃) δ 2.06 (s, 3H), 2.39 (s, 6H), 5.17 (s, 2H), 7.02(d, 2H), 7.12 (dd, 1H).

(2,6-Diethylphenyl)methanol

A mixture of 2,6-diethylbenzyl acetate (2.11 g, 10.2 mmol), MeOH (20mL), H₂O (6 mL), and NaOH (1.99 g, 50 mmol, 5 eq) was stirred at RTovernight. After concentrating, the mixture was extracted with heptane(50 mL). The organic layer was dried (Na₂SO₄), filtered, and evaporatedto give 1.90 g of title product. ¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 1H,OH), 1.24 (t, 6H), 1.37 (br s, 1H), 2.79 (q, 4H), 4.75 (d, 2H), 7.09 (d,2H), 7.21 (dd, 1H). The following compound was prepared by makingappropriate substitutions to the above procedure:(2,6-Dimethylphenyl)methanol. ¹H NMR (300 MHz, CDCl₃) δ 1.25 (t, 1H,OH), 2.41 (s, 6H), 4.72 (d, 2H), 7.0-7.15 (m, 3H).

Part 2: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzoate Ester From a Halo-Substituted Benzoate Ester.

Methyl 2,4-diethylbenzoate

To a mixture of Fe(acac)₃ (0.34 g, 0.96 mmol), methyl2,4-dichlorobenzoate (4.0 g, 19.6 mmol), and N-methyl-2-pyrrolidinone (8mL) in THF (100 mL) at −20° C. under nitrogen was added atetrahydrofuran (THF) solution (1.0 M) of ethylmagnesium bromide (40.0mL, 40.0 mmol) over a period of −5 min. The resulting mixture wasstirred while gradually warming to ambient temperature. Stirring wascontinued for an additional 17 h. The reaction mixture was partitionedbetween water and dichloromethane. The organic layer was separated,washed with brine, dried (MgSO₄), and pumped to dryness under reducedpressure. The brown residue was purified by column chromatography (SiO₂,20% EtOAc/heptane) to give 1.2 g of the desired product as a clear oil(yield of 32%), along with 1.1 g of methyl 4-ethylbenzoate. ¹H NMR (300MHz, DMSO-d₆) δ 1.15 (t, 3H), 1.20 (t, 3H), 2.66 (q, 2H), 2.92 (q, 2H),3.82 (s, 3H), 7.15 (d, 1H), 7.23 (s, 1H), 7.76 (d, 1H).

Part 3: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzaldehyde from Alkyl-Substituted Benzene.

2,5-Diisopropylbenzaldehyde

In a 200 mL single neck RBF, equipped with magnetic stir bar,1,4-diisopropylbenzene (4 g, 25 mmol) was dissolved into 50 mL ofchloroform. To this solution was added SnCl₄ (11.5 g, 5.2 mL, 4.5 mmol)via syringe over 5 min. The reaction mixture was stirred for 5 min andthen Cl₂CHOMe (2.8 g, 24 mmol) was added via syringe over 15 min. Thereaction mixture was stirred for 20 h (RT) and reaction progress wasfollowed by GC/MS. Reaction was quenched by adding 70 mL of water andstirring the mixture for 10 min. Organic layer was separated and washedwith 3 N HCl (2×50 mL). The organic layer was dried over MgSO₄, filteredand concentrated to give 4 g of crude product. This was subjected tocolumn chromatography using 80 g of silica and from 100% heptane to 95:5Heptane:EtOAc as a mobile phase to give 2.4 g of product (yield of 56%).¹H NMR (300 MHz, CDCl₃) δ 1.17 (d, 6H), 1.25 (d, 6H), 2.9 (septet, 1H),4.9 (septet, 1H), 7.4 (m, 2H), 7.65 (d, 1H), 10.35 (s, 1H).

The following compound was prepared based on the above procedure:3,4-Diethylbenzaldehyde. ¹H NMR (300 MHz, DMSO-d₆) δ 1.21 (m, 6H), 2.72(q, 4H), 7.41 (d, 1H), 7.71 (d, 1H), 7.75 (s, 1H).

Part 4: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzyl Alcohol from an Alkyl-Substituted Benzaldehyde.

(2,5-Diisopropylphenyl)methanol

In a 2-neck RBF equipped with magnetic stir bar and N₂ inlet,2,5-diisopropylbenzaldehyde (1.7 g, 9 mmol) was dissolved into 30 mL ofEtOH and NaBH₄ (0.37 g, 10 mmol) was added over 20 min (portion-wise).After 18 h stirring at RT, ˜95% of solvent was removed on a rotaryevaporator and then 5 mL of 0.5 M HCl was added and product wasextracted with 25 mL of EtOAc. Organic layer was washed with 15 mL ofH₂O and 15 mL of brine, dried over Na₂SO₄, filtered and concentrated todryness to obtain crude product. This material was subjected tochromatography using heptane: EtOAc as mobile phase to provide 1.1 g ofpure product. Yield 65%. ¹H NMR (300 MHz, CDCl₃) δ 1.2-1.3 (d, 12H), 2.8(septet, 1H), 3.2 (septet, 1H), 5.7 (s, 2H), 7.1-7.3 (m, 3H).

Part 5: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzyl Alcohol from a Dibromobenzyl Alcohol.

(3,5-Diethylphenyl)methanol

To a cooled (dry ice) mixture of 3,5-dibromobenzyl alcohol (1 g, 3.8mmol) and PdCl₂(dppf) [0.07 eq] in dry THF (10 mL) was added 1.1 M Et₂Zn(15 mL, 16 mmol, 4.4 eq). The resulting mixture was allowed to warm toRT, stirred at 45° C. (programmed block temperature, overnight). Tobring the reaction to completion (disappearance of both startingmaterial and monoalkylated product) additional 1.1 M Et₂Zn (10 mL, 11mmol, 2.9 eq) was added with continued stirring at 45° C. (againovernight). After cooling, the reaction mixture was then added to astirred mixture of dilute HCl and heptane/EtOAc (2:1; ˜200 mL), and theorganic layer was dried (Na₂SO₄), filtered, and evaporated.Chromatography (10% EtOAc/heptane) gave 0.33 g (yield of 53%) of titleproduct. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, 6H), 1.65 (br s, 1H), 2.61(q, 4H), 4.66 (s, 2H), 6.95-7.05 (m, 3H).

The following compounds were prepared by making the appropriatesubstitutions to the above procedure.

(2,5-Diethylphenyl)methanol

¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, 6H), 1.50 (br s, 1H), 2.61 (q, 2H),2.65 (q, 2H), 4.70 (br s, 1H), 7.0-7.2 (m, 3H).

(3,4-Diethylphenyl)methanol

¹H NMR (300 MHz, DMSO-d₆) δ 1.14 (t, 3H), 1.20 (t, 3H), 2.62-2.70 (m,4H), 4.46 (d, 2H), 5.03 (t, 1H), 7.05-7.13 (m, 3H).

Part 6: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzyl Alcohol from an Alkyl-Substituted Benzyl Ester.

(2,4-Diethylphenyl)methanol

To a suspension of lithium aluminum hydride (0.47 g, 11.8 mmol) inanhydrous THF (20 mL) at room temperature under nitrogen was added asolution of methyl 2,4-diethylbenzoate (1.5 g, 7.8 mmol) in THF (15 mL)with stirring over a period of −5 min. The resulting mixture was stirredat room temperature for 60 min and was then quenched by slow addition ofethyl acetate (until bubbling stopped). The mixture was partitionedbetween ethyl acetate and 1N HCl (aq). The aqueous layer was separatedand extracted with ethyl acetate. Organic layers were combined, dried(MgSO4), and evaporated to dryness to give 1.3 g of the desired alcoholas a clear oil (yield of 100%). ¹H NMR (300 MHz, CDCl₃) δ 1.13 (t, 3H),1.17 (t, 3H), 2.58 (q, 2H), 2.61 (q, 2H), 4.43 (d, 2H), 5.02 (t, 1H),7.05-7.11 (m, 3H).

Part 7: Representative Procedure for the Synthesis of anAlkyl-Substituted Benzylhalide from an Alkyl-Substituted Benzyl Alcohol.

2-(Chloromethyl)-1,3-diethylbenzene

To a mixture of (2,6-diethylphenyl)methanol (1.83 g, 11.1 mmol), toluene(20 mL) and DMF (6 drops) was added SOCl₂ (2.1 g, 1.6 eq). The resultingsolution was stirred at RT (1 h). After evaporating to dryness, theresidue was taken up in heptane (−50 mL) and washed with water (−5 mL),dried (Na₂SO₄), filtered and evaporated to give 1.97 g (yield of 97%) oftitle product. ¹H NMR (300 MHz, CDCl₃) δ 1.28 (t, 6H), 2.79 (q, 2H),4.70 (s, 2H), 7.09 (d, 2H), 7.20 (m, 1H).

The following compounds were prepared by making the appropriatesubstitutions to the above procedure.

1-(Chloromethyl)-2,3-dimethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 2.29 (s, 3H), 2.31 (s, 3H), 4.60 (s, 2H),7.0-7.2 (m, 3H).

1-(Iodomethyl)-2,4-dimethyl benzene

¹H NMR (300 MHz, CDCl₃) δ 2.23 (s, 6H), 4.35 (s, 2H), 6.9-7.05 (m, 2H),7.1 (d, 1H).

1-(Chloromethyl)-3,4-dimethyl benzene

¹H NMR (300 MHz, CDCl₃) δ 2.20 (s, 6H), 4.67 (s, 2H), 7.1-7.2 (m, 3H).

2-(Chloromethyl)-1,3-dimethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 2.42 (s, 6H), 4.64 (s, 2H), 7.02 (d, 2H), 7.12(dd, 1H).

2-(Iodomethyl)-1,4-dimethyl benzene

¹H NMR (300 MHz, CDCl₃) δ 2.22 (s, 3H), 2.26 (s, 3H), 4.15 (s, 2H),6.9-7 (m, 2H), 7.16 (d, 1H).

1-(Chloromethyl)-2-ethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 1.2-1.3 (t, 3H), 2.75 (q, 2H), 4.55 (s, 2H),7.1-7.35 (m, 4H).

1-(Chloromethyl)-2,4-diethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 1.27 (m, 6H), 2.69 (q, 2H), 2.78 (q, 2H), 4.66(s, 2H), 6.98-7.10 (m, 2H), 7.26 (m, 1H, contains chloroform signal).

1-(Chloromethyl)-3,4-diethylbenzene

¹H NMR (300 MHz, DMSO-d₆) δ 1.21 (t, 3H), 1.25 (t, 3H), 2.60 (q, 2H),2.64 (q, 2H), 4.71 (s, 2H), 7.15-7.40 (m, 3H).

1-(Chloromethyl)-3,5-diethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 6H), 2.63 (q, 4H), 4.54 (s, 2H), 7.01(d, 2H), 7.05 (d, 1H).

2-(Chloromethyl)-1,4-diethylbenzene

¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, 3H), 1.26 (t, 3H), 2.61 (q, 2H), 2.84(q, 2H), 4.60 (s, 2H), 7.0-7.2 (m, 3H).

2-(Bromomethyl)-1,4-diisopropylbenzene

¹H NMR (300 MHz, CDCl₃) δ 1.1-1.2 (d, 6H), 1.2-1.3 (d, 6H), 2.8 (septet,1H), 3.25 (septet, 1H), 4.55 (s, 2H), 7.1-7.3 (m, 3H).

1-(Chloromethyl)-4-ethylbenzene and 1-(Chloromethyl)-4-isopropylbenzeneExample 5 Representative Procedures for the Synthesis ofBenzo[e][1,4]diazepin-2(3H)-ones from Imidoyl Chlorides

Part I: Palladium-Coupling Reaction.

(Z)-7-Chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one(E1)

In a 1 L 3-neck RBF equipped with magnetic stir bar, condenser,thermocouple, and N₂ inlet, crude D1 (30 g, 0.124 mol) was dissolvedinto 300 mL of ethylene glycol dimethyl ether (DME). To this was added asolution of Na₂CO₃ (21 g, 0.2 mol in 200 mL of H₂O) followed by additionof 4-methoxyphenyl boronic acid (22 g, 0.145 mol) and Pd(PPh₃)₄ (1.2 g,8.3 mmol). The reaction mixture was heated in a 85° C. oil bath, underN₂, for 2 h and then cool to room temp. To this was added 200 mL ofEtOAc and the mixture stirred for 5 min. The organic layer was separatedand washed with H₂O (200 mL) and brine (200 mL). The organic layer wasdried over MgSO₄ and then concentrated to dryness to give 53 g of crudeproduct. This material was subjected to silica gel chromatography using210 g of silica gel and EtOAc/hepatene (12:88 to 30:70 to 50:50 to70:30; total of 8 L mobile phase). Fractions containing pure productwere combined and concentrated to dryness to give 42.7 g of pure productin approximately quantitative yield. ¹H NMR (300 MHz, CDCl₃) δ 3.38 (s,3H), 3.73 (d, 1H), 3.85 (s, 3H), 4.75 (d, 1H), 6.9 (m, 2H), 7.31 (m,2H), 7.48-7.58 (m, 3H).

(Z)-7-Chloro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one(E2)

In a 1 L 3-neck RBF equipped with magnetic stir bar, condenser,thermocouple, and N₂ inlet, crude D2 (54 g) was dissolved into 360 mL ofDME. To this was added a solution of Na₂CO₃ (23 g, 0.15 mol, in 250 mLof H₂O) followed by the addition of 4-methoxyphenyl boronic acid (22.7g, 0.15 mol) and Pd(PPh₃)₄ (1.4 g, 1.2 mmol). The reaction mixture washeated in a 85° C. oil bath for 2 h and then cooled (RT). To this wasadded 200 mL of EtOAc and the mixture was stirred for 5 min. The organiclayer was separated and washed with 200 mL H₂O and then brine. Theorganic layer was concentrated to dryness to give 68 g of crude product.This material was subjected to column chromatography using 550 g ofsilica gel and 25/75 to 60/40 EtOAc/heptane. Fractions containing pureproduct were combined to give 21 g of pure product, and other fractionscontaining a small amount of impurity (by TLC) gave another 20 g ofproduct. ¹H NMR spectra of both lots appeared identical. A total of 41 gof product was obtained, providing the product in 72% yield over twosteps. ¹H NMR (300 MHz, CDCl₃) δ 3.7 (s, 3H), 3.80 (d, 1H), 3.85 (s,3H), 4.57 (d, 1H), 4.85 (d, 1H), 5.57 (d, 1H), 6.63 (d, 2H), 6.85-6.95(m, 4H), 7.16 (d, 1H), 7.3-7.44 (m, 4H).

Part II: C3-Alkylation of Benzo[e][1,4]diazepin-2(3H)-ones.

(Z)-7-Chloro-5-(4-methoxyphenyl)-1-methyl-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

To a stirred and cooled (dry ice/acetone bath) solution of E1 (0.50 g,1.59 mmol) in THF (8 mL) was slowly added 1 M KO^(t)Bu (2.4 mL, 2.4mmol, 1.5 eq). The resulting deep red mixture was stirred over dryice/acetone bath ˜10 min followed by the slow addition of a solution of2-methylbenzyl bromide (0.46 g, 2.5 mmol, 1.5 eq) in THF (2 mL). Afterstirring another ˜35 min at −78° C., the reaction mixture was quenchedwith water and diluted with EtOAc. The organic layer was dried (Na₂SO₄),filtered and evaporated (rotovap, then high vacuum). Chromatography oversilica gel using 20-40% EtOAc/heptane gave 0.56 g (yield of 84%) of thetitle product. Noting that, when using benzyl chlorides as alkylatingagents, tetrabutyl ammonium iodide was added along with the alkylatingagent at the low temperature. ¹H NMR (300 MHz, CDCl₃) δ 2.38 (s, 3H),3.41 (s, 3H), 3.5-3.65 (m, 2H), 3.74 (dd, 1H), 3.84 (s, 3H), 6.89 (dt,2H), 7.05-7.15 (m, 3H), 7.25-7.35 (m, 3H), 7.45-7.5 (m, 3H).

The following compounds were prepared by making the appropriatesubstitutions to the above procedures.

(Z)-7-Chloro-5-(4-methoxyphenyl)-3-(3-bromobenzyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 3.40 (s, 3H), 3.45-3.55 (m, 2H), 3.67 (dd,1H), 3.86 (s, 3H), 6.91 (d, 2H), 7.14 (t, 1H), 7.25-7.3 (m, 3H), 7.33(ddd, 1H), 7.45-7.55 (m, 4H).

(Z)-7-Chloro-5-(4-methoxyphenyl)-3-(3-methylbenzyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 2.31 (s, 3H), 3.38 (s, 3H), 3.5-3.6 (m, 2H),3.68 (dd, 1H), 3.82 (s, 3H), 6.88 (d, 2H), 6.99 (m, 1H), 7.1-7.2 (m,3H), 7.2-7.3 (m, 3H+CHCl₃), 7.4-7.53 (m, 3H).

(Z)-7-Chloro-3-(2,6-dimethylbenzyl)-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 2.39 (s, 6H), 3.40 (dd, 1H), 3.41 (s, 3H),3.72 (dd, 1H), 3.82 (s, 3H), 3.87 (dd, 1H), 6.87 (d, 2H), 6.98-7.04 (m,3H), 7.22 (fine d, 1H), 7.28 (d, 1H), 7.39 (d, 2H), 7.48 (dd, 1H).

(Z)-7-Chloro-3-(3,5-dimethylbenzyl)-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 2.28 (s, 6H), 3.39 (s, 3H), 3.47 (m, 2H), 3.65(m, 1H), 3.85 (s, 3H), 6.82 (s, 1H), 6.89 (m, 2H), 6.97 (s, 2H), 7.26(m, 2H), 7.47 (m, 3H).

(Z)-3-(2,6-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.13 (t, 6H), 2.65-2.8 (m, 2H), 2.8-2.95 (m,2H), 3.43 (s, 3H), 3.49 (dd, 1H), 3.68 (dd, 1H), 3.8-3.95 (m, 4H,includes singlet for OMe at 3.84), 6.87 (d, 2H), 7.04 (dd, 2H), 7.13(dd, 1H), 7.20 (fine d, 1H), 7.28 (d, 1H), 7.40 (d, 2H), 7.47 (dd, 1H).

(Z)-3-(3,4-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.12 (m, 6H), 2.45-2.75 (m, 4H; containsDMSO signal), 3.3-3.45 (m, 2H; contains benzylic protons, N-Me, and H₂Osignals), 3.69 (m, 1H), 3.81 (s, 3H), 6.99 (m, 3H), 7.09 (m, 1H), 7.21(s, 2H), 7.48 (d, 2H), 7.62 (d, 1H), 7.70 (fine dd, 1H).

(Z)-3-(3,5-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.21 (t, 6H), 2.58 (q, 4H), 3.40 (s, 3H), 3.52(d, 2H), 3.69 (t, 1H), 3.84 (s, 3H), 6.85-6.95 (m, 3H; contains d at6.90), 7.02 (fine d, 2H), 7.2-7.3 (m, 2H; overlaps with CHCl₃ signal),7.47 (dd, 1H), 7.53 (d, 2H).

(Z)-3-(4-Isopropylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.23 (d, 6H), 2.84 (septet, 1H), 3.39 (s, 3H),3.51 (m, 2H), 3.69 (dd, 1H), 3.85 (s, 1H), 6.89 (m, 2H), 7.12 (m, 2H),7.26 (m, 4H), 7.44-7.54 (m, 3H).

(Z)-3-(2,5-Diisopropylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 0.96 (d, 3H), 1.1 (d, 3H), 1.14 (d, 3H),1.16 (d, 3H), 2.8 (septet, 1H), 3.17 (septet, 1H), 3.35 (s, 3H), 3.40(m, 2H), 3.70 (m, 1H), 3.80 (s, 3H), 6.9-7.2 (m, 6H), 7.45 (d, 2H), 7.6(d, 1H), 7.7 (d, 1H).

(Z)-3-(3-Bromobenzyl)-1-(4-methoxybenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]-diazepin-2(3H)-one

To a stirred and cooled (dry ice/acetone bath) solution of E2 (6.00 g,14.2 mmol) in THF (80 mL) was slowly added 1 M KO^(t)Bu (21 mL, 21 mmol,1.5 eq). The resulting deep red mixture was stirred over dry ice/acetonebath ˜10 min followed by the slow addition of a solution of3-bromobenzyl bromide (5.10 g, 21.4 mmol, 1.5 eq) in THF (15 mL). Afterstirring another ˜45 min at −78° C., the reaction mixture was quenchedwith saturated brine and diluted with EtOAc. The organic layer was dried(Na₂SO₄), filtered and evaporated (rotovap, then high vacuum).Chromatography over silica gel using 10-30% EtOAc/heptane gave 7.08 g(yield of 84%) of the title product. Noting that when using benzylchlorides as alkylating agents, tetrabutyl ammonium iodide was addedalong with the alkylating agent at low temperature. ¹H NMR (300 MHz,CDCl₃) δ 3.57 (d, 2H), 3.70 (s, 3H), 3.73 (t, 1H), 3.83 (s, 3H), 4.59(d, 1H), 5.62 (d, 1H), 6.61 (d, 2H), 6.8-6.9 (m, 4H), 7.2-7.45 (m, 8H),7.63 (fine d, 1H).

The following compounds were prepared by making the appropriatesubstitutions to the above procedures.

(Z)-3-(2,6-Dimethylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 2.41 (s, 6H), 3.50 (dd, 1H), 3.70 (s, 3H),3.79 (dd, 1H), 3.84 (s, 3H), 3.90 (dd, 1H), 4.58 (d, 1H), 5.69 (d, 1H),6.62 (d, 2H), 6.83 (d, 2H), 6.90 (d, 2H), 6.95-7.03 (m, 3H), 7.06 (fined, 1H), 7.16 (d, 2H), 7.32 (d, 1H), 7.37 (dd, 1H).

(Z)-3-(3,5-Dimethylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.2 (s, 6H), 3.3 (m, 2H), 3.6 (s, 3H), 3.7(m, 1H), 3.85 (s, 3H), 5.8 (d, 1H), 6.45 (d, 1H), 6.65 (d, 2H), 6.85 (m,3H), 6.95 (m, 3H), 7.1 (s, 1H), 7.2 (d, 2H), 7.6 (d, 1H), 7.7 (d, 1H).

(Z)-3-(3,4-Dimethylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.10 (s, 6H; 2 methyl groups), 3.39 (m, 2H,contains benzylic protons and H₂O signal), 3.68 (s, 3H), 3.81-3.89 (m,4H, contains N-PMB-OMe signal), 4.99 (d, 1H), 5.41 (d, 1H), 6.71 (d,2H), 6.88 (d, 2H), 7.0-7.2 (m, 6H), 7.31 (d, 2H), 7.65 (m, 2H)

(Z)-3-(2,6-Diethylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.18 (t, 6H), 2.65-2.95 (m, 4H), 3.59 (dd,1H), 3.70 (s, 3H), 3.74 (dd, 1H), 3.83 (s, 3H), 3.90 (dd, 1H), 4.58 (d,1H), 5.71 (d, 1H), 6.62 (d, 2H), 6.82 (d, 2H), 6.89 (d, 2H), 7.0-7.1 (m,3H), 7.1-7.2 (m, 3H), 7.32 (d, 1H), 7.36 (dd, 1H).

(Z)-3-(3,4-Diethylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo-[e][1,4]diazepin-2(3H)-one(Z)-3-(3,5-Diethylbenzyl)-1-(4-methoxybenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.21 (t, 6H), 2.59 (q, 4H), 3.5-3.65 (m, 2H),3.70 (s, 3H), 3.76 (dd, 1H), 3.85 (s, 3H), 6.61 (d, 2H), 6.8-6.95 (m,5H), 7.04 (fine d, 2H), 7.10 (fine d, 1H), 7.2-7.28 (m, 2H; overlapswith CHCl₃ signal), 7.30 (d, 1H), 7.37 (dd, 1H).

(Z)-3-(4-Isopropylbenzyl)-7-chloro-1-(4-methoxybenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.15 (d, 6H), 2.85 (septet, 1H), 3.25-3.45(m, 3H), 3.65 (s, 3H), 3.75-3.85 (m, 4H), 4.80 (d, 1H), 5.45 (d, 1H),6.60 (m, 2H), 6.85 (m, 2H), 6.95 (m, 2H), 7.0-7.3 (m, 6H), 7.63 (dd,1H), 7.74 (d, 1H).

(Z)-3-(2,5-Diisopropylbenzyl)-1-(4-methoxybenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.1 (d, 3H), 1.2 (d, 3H), 1.25 (m, 6H), 2.87(septet, 1H), 3.2 (septet, 1H), 3.6-3.7 (m, 5H), 3.8 (m, 1H), 3.9 (s,3H), 4.55 (d, 1H), 6.7 (d, 1H), 6.58 (m, 2H), 6.8 (m, 2H), 6.9 (m, 2H),7.0-7.4 (m, 8H).

7-Chloro-5-(4-fluorophenyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

7-Chloro-5-(4-fluorophenyl)-1-(4-methoxybenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one(90 mg, 0.220 mmol) was dissolved in dry THF (2 mL) and cooled to −78°C. under nitrogen. Potassium tert-butoxide (49 mg, 0.437 mmol) was addedas a solid in one portion and the resulting red solution was stirredvigorously for 5 min. To this solution was added 2-methylbenzyl bromide(25 μL, 0.262 mmol) by syringe. The mixture was stirred for 1 h at −78°C., then the cold bath was removed. After another 1 h (warming to roomtemperature), the reaction was quenched with MeOH and diluted with ethylacetate. The organic layer was washed twice with water and once withbrine, and then dried over MgSO₄. Chromatography on silica gel elutingwith 100% hexanes to 30% ethyl acetate in hexanes provided7-chloro-5-(4-fluorophenyl)-1-(4-methoxybenzyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one(44 mg, 39%). APCI MS m/z 535.2 [M+Na⁺], 513.3 [M⁺]; ¹H NMR (300 MHz,CDCl₃) δ 7.43-6.98 (m, 11H), 6.88 (d, J=9 Hz, 2H), 6.61 (dd, J=7 Hz, 2Hz, 2H), 5.69 (d, J=15 Hz, 1H), 4.55 (d, J=15 Hz, 1H), 3.85 (t, J=7 Hz,1H), 3.70 (s, 3H), 3.63 (d, J=7 Hz, 2H), 2.37 (s, 3H).

Example 6

(Z)-7-Chloro-3-(3-ethylbenzyl)-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

To a stirred and cooled (dry ice-acetone bath) solution of ArBr (1.21 g,2.5 mmol) and PdCl₂(dppf) [0.22 g] in dry THF (10 mL) was added 1 MEt₂Zn (9.3 mL, 10 mmol, 4 eq). After warming to RT, the reaction mixturewas stirred at 50° C. until HPLC indicated reaction to be complete.After aqueous workup, chromatography gave 0.95 g (yield of 88%) of titleproduct. ¹H NMR (300 MHz, CDCl₃) δ 1.22 (t, 3H), 2.62 (q, 2H), 3.55 (d,2H), 3.70 (t, 1H), 3.84 (s, 3H), 6.89 (d, 2H), 7.03 (m, 1H), 7.1-7.3 (m,5H), 7.4-7.55 (m, 3H).

The following compounds were made by making the appropriatesubstitutions to the above procedure.

(Z)-7-Chloro-3-(3-ethylbenzyl)-1-(4-methoxybenzyl)-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 3H), 2.63 (q, 2H), 3.55-3.65 (m, 2H),3.68 (s, 3H), 3.80 (dd, 1H), 3.83 (s, 3H), 4.59 (d, 1H), 5.63 (d, 1H),6.61 (d, 2H), 6.85 (d, 2H), 6.88 (d, 2H), 7.04 (dt, 1H), 7.10 (fine d,1H), 7.13-7.26 (m, 5H), 7.30 (d, 1H), 7.37 (dd, 1H).

(Z)-7-Chloro-3-(3-isopropylbenzyl)-5-(4-methoxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.24/1.25 (2 overlapping doublets, 6H), 2.88(heptet, 1H), 3.38 (s, 3H), 3.5-3.6 (m, 2H), 3.69 (t, 1H), 3.84 (s, 3H),6.89 (d, 2H), 7.07 (dt, 1H), 7.13 (dt, 1H), 7.15-7.3 (m, 4H; includesCHCl₃ singlet), 7.47 (dd, 1H), 7.52 (d, 2H).

Example 7 Representative Procedures for Removal of a p-MethoxybenzylGroup

Method A

(Z)-3-(3-Ethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

To a solution of N1-PMB-protected benzodiazepinone (1 g) in MeCN (17 mL)and H₂O (3 mL) was added cerium(IV) ammonium nitrate (7 g). Theresulting mixture was stirred until TLC showed reaction to be completeand was then diluted with water, EtOAc and heptane. The organic layerwas dried (Na₂SO₄), filtered and evaporated to a crude solid.Chromatography using increasing amounts of DCM/EtOAc (1:1) in heptane(up to 25:25:50 DCM/EtOAc/heptane) gave 0.55 g (yield of 57%) of titleproduct. ¹H NMR (300 MHz, DMSO-d₆) δ 1.19 (t, 3H), 2.57 (q, 2H), 3.2-3.4(m, 2H), 3.7-3.8 (m, 4H; contains OMe singlet at 3.79), 7.0-7.1 (m, 3H),7.1-7.35 (m, 5H), 7.39 (d, 2H), 7.71 (dd, 1H), 10.9 (br s, 1H).

The following compounds were made by making the appropriatesubstitutions to the above procedure.

(Z)-3-(2,5-Dimethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.21 (s, 3H), 2.28 (s, 3H), 3.23-3.37 (m,2H), 3.63 (m, 1H), 3.85 (s, 3H), 6.86-7.4 (m, 9H), 7.55 (dd, 1H), 10.65(s, 1H).

Z)-3-(3,5-Dimethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.25 (s, 6H), 3.25 (m, 2H), 3.85 (m., 4H),6.8-7.8 (m, 10H), 10.9 (s, 1H).

(Z)-3-(4-Isopropylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.15 (d, 6H), 2.85 (septet, 1H), 3.25-3.38(m, 2H), 3.8 (br s, 4H), 6.95-7.50 (m, 10H), 7.73 (dd, 1H), 10.9 (s,1H). Method B

(Z)-3-(2,6-Dimethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

A mixture of PMB-protected benzodiazepinone (0.78 g, 1.45 mmol), anisole(25 mL), and AlBr₃ (3.8 g) was stirred at 85° C. (1 h), allowed to cool,then treated with ice and diluted with EtOAc and heptane. The organiclayer was removed, dried (Na₂SO₄), filtered, concentrated to a volume of˜2 mL, and chromatographed (step-wise gradient up to 50% EtOAc/heptane)to give the title product in 89% yield. ¹H NMR (300 MHz, CDCl₃) δ 2.42(s, 6H), 3.43 (dd, 1H), 3.65-3.9 (m, 5H; contains OMe singlet at 3.82),6.88 (d, 2H), 7.0-7.1 (m, 4H), 7.2-7.3 (m, 1H; overlaps with CHCl₃signal), 7.32 (d, 2H), 7.43 (dd, 1H), 7.9 (s, 1H).

The following compounds were made by making the appropriatesubstitutions to the above procedure.

(Z)-3-(2,6-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.20 (t, 6H), 2.7-3.0 (m, 4H), 3.51 (dd, 1H),3.68 (dd, 1H), 3.75-3.9 (m, 4H; contains OMe singlet at 3.83), 6.86 (d,2H), 7.0-7.2 (m, 4H), 7.2-7.3 (m, 1H; overlaps with CHCl₃ signal), 7.32(d, 2H), 7.43 (dd, 1H), 7.96 (br s, 1H).

(Z)-3-(3,4-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.12 (m, 6H), 2.45-2.75 (m, 4H; containsDMSO signal), 3.2-3.35 (m, 2H; contains benzylic protons and H₂Osignals), 3.65 (m, 1H), 3.80 (s, 3H), 6.99 (d, 2H), 7.09 (m, 1H), 7.15(s, 1H), 7.21-7.27 (m, 2H), 7.38 (d, 2H), 7.62 (fine dd, 1H), 10.71 (s,1H).

(Z)-3-(3,5-Diethylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, CDCl₃) δ 1.23 (t, 6H), 2.60 (q, 4H), 3.51 (d, 2H), 3.72(t, 1H), 3.83 (s, 3H), 6.85-6.95 (m, 3H), 7.0-7.1 (m, 3H), 7.29 (fine d,1H), 7.4-7.5 (m, 3H), 8.32 (br s, 1H).

(Z)-3-(2,5-Diisopropylbenzyl)-7-chloro-5-(4-methoxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

MS, m/z=475.2 [M+1].

Method C

7-Chloro-5-(4-fluorophenyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

7-Chloro-5-(4-fluorophenyl)-1-(4-methoxybenzyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one(44 mg, 0.086 mmol) was dissolved in dichloroethane (1 mL) undernitrogen and 200 L of anisole was added followed by AlCl₃ (69 mg, 0.515mmol). This mixture was heated to 85° C. for 2.5 h then allowed to cooland diluted with ethyl acetate and ice. This biphasic mixture wasstirred vigorously for 20 min, then partitioned, and the organic layerwas washed with water twice, then brine once. The aqueous layers wereback-extracted once with ethyl acetate, and the combined organicextracts dried over MgSO₄. Chromatography on silica gel (5%-30%-40%-50%ethyl acetate in hexanes) provided the title compound (27 mg, 80%) as ayellow solid. APCI MS m/z 415.1 [M+Na⁺], 393.1 [M+H⁺]; ¹H NMR (300 MHz,CDCl₃) δ 9.28 (s, 1H), 7.61-7.16 (m, 11H), 3.91 (t, J=7 Hz, 1H),3.72-3.69 (m, 2H), 2.50 (s, 3H).

Example 8 Representative Procedure for O-Demethylation of ap-Methoxybenzyl Group(Z)-7-Chloro-5-(4-hydroxyphenyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

To a solution of ArOMe precursor (0.6 g, 1.4 mmol) in CH₂Br₂ (20 mL) wasadded EtSH (7 mL) and then AlBr₃ (1.7 g, 6.3 mmol, 4.5 eq). Theresulting mixture was stirred overnight and then treated with ice (20 g)and after one hour filtered. The resulting solid was triturated with 50%DCM/heptane and then vacuum dried to give 445 mg (yield of 80%) of thetitle product as a light yellow solid. ¹H NMR (300 MHz, DMSO-d₆) δ 2.31(s, 3H), 3.25-3.45 (m, 2H), 3.73 (dd, 1H), 6.80 (d, 2H), 7.02-7.18 (m,3H), 7.2-7.3 (m, 5H), 7.64 (dd, 1H), 10.0 (br s, 1H), 10.7 (br s, 1H).MS, m/z 391.7 [M+1]

The following compounds were made by making the appropriatesubstitutions to the above procedure.

(Z)-7-Chloro-5-(4-hydroxyphenyl)-3-(2-methylbenzyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.32 (s, 3H), 3.3-3.5 (m, 5H; containssinglet for NMe at 3.36), 3.90 (t, 1H), 6.87 (d, 2H), 7.05-7.15 (m, 3H),7.20 (m, 1H), 7.29 (fine d, 1H), 7.35 (d, 2H), 7.65 (d, 1H), 7.78 (dd,1H), 9-11 (br s, 1H). MS, m/z 405.3 [M+1].

(Z)-3-(2,3-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.18 (d, 6H; 2 methyl groups), 3.35-3.5 (m,2H; contains H₂O signal), 3.65 (t, 1H), 6.78 (d, 2H), 6.98 (d, 2H), 7.09(t, 1H), 7.22-7.27 (m, 4H), 7.62 (dd, 1H), 9.92 (br s, 1H), 10.7 (s,1H). MS, m/z 405.2 [M+1]

(Z)-3-(2,6-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.32 (s, 6H), 3.21 (dd, 1H), 3.55-3.7 (m,2H), 6.77 (d, 2H), 6.98 (s, 3H), 7.13 (d, 2H), 7.18 (fine d, 1H), 7.26(d, 1H), 7.60 (dd, 1H), 9.92 (br s, 1H), 10.7 (s, 1H); MS, m/z 405.2[M+1].

(Z)-3-(2,6-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-11H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.31 (s, 6H), 3.20 (dd, 1H), 3.33 (s, 3H),3.6-3.7 (m, 2H), 6.78 (d, 2H), 6.96 (s, 3H), 7.18 (fine d, 1H), 7.22 (d,2H), 7.60 (d, 1H), 7.68 (dd, 1H), 9.97 (s, 1H); MS, m/z 419.2 [M+1].

(Z)-3-(3,5-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.33 (s, 6H), 3.1-3.35 (m, 3H), 3.55 (m,1H), 6.65 (m, 3H), 6.90 (d, 2H), 7.30-7.40 (m, 4H), 7.60 (dd, 1H), 9.95(s, 1H), 10.63 (s, 1H); MS, m/z 405.2 [M+1].

(Z)-3-(3,5-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆), δ 2.20 (s, 6H), 3.24-3.31 (m, 5H), 3.61 (m,1H), 6.78-6.90 (m, 5H), 7.22 (d, 1H), 7.35 (m, 2H), 7.57 (d, 1H), 7.67(dd, 1H), 9.98 (s, 1H); MS, m/z 419.3 [M+1].

(Z)-3-(3,4-Dimethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 2.18 (fine d, 6H), 3.15-3.45 (m, 2H;contains water signal), 3.59 (t, 1H), 6.80 (d, 2H), 7.01 (s, 2H), 7.08(s, 1H), 7.22 (m, 2H), 7.28 (d, 2H), 7.59 (fine dd, 1H), 9.98 (br s,1H), 10.65 (s, 1H); MS, m/z 405.8 [M+1].

(Z)-3-(2,6-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.09 (t, 6H), 2.6-2.9 (m, 4H), 3.27 (m,overlaps with H₂O signal), 3.51 (dd, 1H), 3.61 (dd, 1H), 6.77 (d, 2H),6.96-7.2 (m, 6H), 7.26 (d, 1H), 7.60 (d, 1H), 9.93 (br s, 1H), 10.7 (s,1H); MS, m/z 433.2 [M+1].

(Z)-3-(2,6-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-11H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.07 (t, 6H), 2.6-2.85 (m, 4H), 3.2-3.4 (m,contains signals for C-3 methine proton, NMe, and H₂O), 3.5-3.7 (m, 2H),6.78 (d, 2H), 6.94-7.02 (m, 2H), 7.08 (dd, 1H), 7.18 (fine d, 1H), 7.23(d, 2H), 7.60 (d, 1H), 7.69 (dd, 1H), 9.97 (s, 1H); MS, m/z 447.3 [M+1].

(Z)-3-(3,4-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.07 (m, 6H), 2.48-2.69 (m, 4H, containsDMSO signal), 3.20-3.45 (m, contains signals for benzylic protons andH₂O), 3.64 (fine dd, 1H), 6.82 (d, 2H), 7.03 (m, 2H), 7.12 (s, 1H),7.2-7.25 (m, 2H), 7.29 (d, 2H), 7.60 (fine dd, 1H), 9.97 (s, 1H), 10.68(s, 1H); MS, m/z 433.3 [M+1].

(Z)-3-(3,4-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-11H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.07 (m, 6H), 2.48-2.59 (m, 4H, containsDMSO signal), 3.25-3.33 (m, contains signals for benzylic protons, NMe,and H₂O), 3.64 (fine dd, 1H), 6.82 (d, 2H), 7.01 (s, 2H), 7.09 (s, 1H),7.23 (fine d, 1H), 7.38 (d, 2H), 7.59 (d, 1H), 7.68 (fine dd, 1H), 9.97(s, 1H); MS, m/z 447.2 [M+1].

(Z)-3-(3,5-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.18 (t, 6H), 2.55 (q, 4H), 3.25-3.4 (m,2H), 3.88 (br t, 1H), 6.85-6.92 (m, 3H), 7.03 (s, 1H), 7.27-7.35 (m,4H), 7.75 (dd, 1H), 10-11 (br s), 11.03 (br s, 1H); MS, m/z 433.3 [M+1].

(Z)-3-(3,5-Diethylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.14 (t, 6H), 2.52 (q, overlaps with DMSOmultiplet), 3.30 (t, 2H), 3.68 (t, overlaps with H₂O signal), 6.78-6.86(m, 3H), 6.96 (fine d, 1H), 7.22 (fine d, 1H), 7.37 (d, 2H), 7.59 (d,1H), 7.69 (dd, 1H), 10.00 (br s, 1H); MS, m/z 447.3 [M+1].

(Z)-3-(4-Isopropylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) 1.15 (d, 6H), 2.82 (septet, 1H), 3.24-3.38 (m,2H), 3.63 (m, 1H), 6.79 (m, 2H), 7.10-7.30 (m, 8H), 7.59 (dd, 1H), 9.45(s, 1H), 10.61 (s, 1H); MS, m/z 419.3 [M+1].

(Z)-3-(4-Isopropylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.15 (d, 6H), 2.84 (septet, 1H), 3.26-3.41(m, 5H), 3.77 (m, 1H), 6.84 (m, 2H), 7.10-7.40 (m, 7H), 7.63 (d, 1H),7.73 (dd, 1H), 10.2 (br s, 1H); MS, m/z 433.3 [M+1].

(Z)-3-(2,5-Diisopropylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 1.02 (d, 3H), 1.12 (d, 3H), 1.17 (d, 3H),1.19 (d, 3H), 2.81 (septet, 1H), 3.18 (septet, 1H), 3.37 (d, 2H), 3.58(t, 1H), 6.74 (d, 2H), 7.04 (dd, 1H), 7.12-7.26 (m, 6H), 7.60 (dd, 1H),9.94 (s, 1H), 10.68 (s, 1H); MS, m/z 461.4 [M+1].

(Z)-3-(2,5-Diisopropylbenzyl)-7-chloro-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

¹H NMR (300 MHz, DMSO-d₆) δ 0.98 (d, 3H), 1.12 (d, 3H), 1.15 (d, 3H),1.18 (d, 3H), 2.80 (septet, 1H), 3.15 (septet, 1H), 3.39 (m, 2H), 3.64(m, 1H), 6.76 (d, 2H), 7.03 (dd, 1H), 7.15 (m, 2H), 7.20 (d, 1H), 7.32(d, 2H), 7.58-7.7 (m, 2H), 9.97 (s, 1H); MS, m/z 475.2 [M+1].

Example 9 Representative Palladium Coupling Procedure for Making aC5-fluorophenyl-benzo[e][1,4]diazepin-2(3H)-one(Z)-7-Chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one

A 10 mL sealed tube was charged with(E)-5,7-dichloro-3-(3,4-diethylbenzyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one(0.25 g, 0.505 mmol, 1 equivalent), 4-fluorophenylboronic acid (0.071 g,0.505 mmol, 1 equivalent), Pd(OAc)₂, (0.2 equivalents), PPh₃ (0.2equivalents), Cs₂CO₃ (2 equivalents) and DMF (3 mL). The tube wasevacuated, flushed with nitrogen gas and placed in an oil bath at 100°C. for 1.5 hour. The mixture was cooled to ambient temperature, dilutedwith ethyl acetate (10 mL) and water (5 mL), and filtered through a padof celite. The layers were separated and the organic phase was washedwith brine (3×10 mL), dried over sodium sulfate, filtered, andconcentrated. The residue was purified by chromatography (silica gel,70:30 hexanes:ethyl acetate) to provide an oil. This oil was dissolvedin a mixture of acetonitrile (3 mL) and water (1 mL), and the solutionwas freeze-dried overnight to give(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one(0.12 g, 43%) as an off-white solid: mp 54-56° C.; ¹H NMR (300 MHz,DMSO-d₆) δ 7.41-7.22 (m, 4H), 7.18-6.98 (m, 6H), 6.87 (d, J=8.6 Hz, 2H),6.60 (d, J=4.6 Hz, 2H), 5.66 (d, J=15 Hz, 1H), 4.56 (d, J=15 Hz, 1H),3.82-3.77 (m, 1H), 3.70 (s, 3H), 3.63-3.55 (m, 2H), 2.67-2.58 (m, 4H),1.26-1.12 (m, 6H); APCI MS m/z 556 [M+H⁺].

Example 10 Representative Procedure for Demethylation Using AluminumChloride(Z)-7-Chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one

To a stirred solution of(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one(0.1 g, 0.18 mmol) in anisole (3 mL) was added aluminum chloride (0.144g, 1.08 mmol). The mixture was heated at 85° C. for 1 hour, cooled toambient temperature and poured into a mixture of ice/water (10 mL) andethyl acetate (20 mL) and stirred vigorously for 10 min. The layers wereseparated and the organic phase was dried over sodium sulfate, filtered,and then concentrated. The residue was purified by chromatography(silica gel, 95:5 CH₂Cl₂:MeOH) to provide an oil which was dissolved ina mixture of acetonitrile (3 mL) and water (1 mL). This solution wasfreeze-dried overnight to give(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-oneas an off-white solid: mp 88-90° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 10.71(s, 1H), 7.64 (dd, J=8.8, 2.5 Hz, 1H), 7.49 (t, J=5.6 Hz, 2H), 7.30-7.23(m, 4H), 7.13-7.01 (m, 3H), 3.65 (t, J=5.5 Hz, 1H), 3.32-3.31 (m, 2H),2.56 (quintet, J=7.3 Hz, 4H), 1.13 (q, J=7.2 Hz, 6H); APCI MS m/z 435[M+H⁺].

Example 11

The compound having chemical name(Z)-7-chloro-3-(2,6-dimethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one(i.e., “compound H”) was evaluated for toxicity and inhibition ofepidermal hyperplasia induced by 14 all-trans retinoic acid (RA).

General Procedures:

Human Skin.

Replicate 6-mm punch biopsies of full-thickness psoriatic plaque skinwere obtained from human skin donors with psoriasis. These biopsies wereused in the transplant studies. Six-mm punch biopsies of sun-protected(hip) skin from non-psoriatic donors were obtained as controls. Inaddition, replicate 2-mm full-thickness punch biopsies of sun-protectedhip skin were obtained from normal donors for use in organ culturestudies.

Human Skin Organ Cultures.

Immediately upon biopsy, the replicate 2-mm punch biopsies(non-psoriatic skin only) were immersed in culture medium consisting ofKeratinocyte Basal Medium (KBM) (Lonza, Walkersville, Md.). KBM is alow-Ca²⁺, serum-free modification of MCDB-153 medium. It wassupplemented with CaCl₂ to bring the final Ca²⁺ concentration to 1.4 mM.Biopsies were incubated in wells of a 24-well dish containing 400 μl ofCa²⁺-supplemented KBM with or without additional treatments (RA and/orCompound H). Cultures were incubated at 37° C. in an atmosphere of 95%air and 5% CO₂. Other than to maintain the tissue in a minimal volume ofmedium, nothing further was done to ensure a strict air-liquidinterface. Incubation was for 8 days, with change of medium and freshtreatments provided every second day. At the end of the incubationperiod, tissue was fixed in 10% buffered formalin and examinedhistologically after staining with hematoxylin and eosin. Routinely, 3-6tissue sections were prepared from each block. Epidermal thicknessmeasurements were made at several sites in each tissue section (distancefrom the dermal-epidermal juncture to the top of the viable portion ofthe epithelium). The organ culture procedure employed here has beendescribed in the past (Varani et al. (1993) Amer. J. Pathol.142:189-198; and Varani et al., (1994) J. Clin. Invest. 94:1747-1753).In order to evaluate the toxicity of compound H, tissue was examinedhistologically and necrosis was assessed on a 0 to 4+ scale, with 0indicating no change from control and 4+ indicating complete necrosis ofthe epidermis (n=6). Scores ≦1 were taken as normal.

Results:

Compound H had no detectable toxicity at a dose of 0.5 μM or 1.0 μM.Slight toxicity was observed at a 2.0 μM dose of compound H. Inhibitionof RA-induced epidermal hyperplasia was measured as a percent reductionin epidermal thickness (n=3). The average epidermal thickness in theabsence of RA was 70-90 μm, while the average epidermal thickness was275 μm in cells exposed to RA. As shown in Table 3, compound H caused areduction in Retinoid-Induced Hyperplasia, as measured by a decreased inthe epidermal thickness compared to cells exposed to RA alone.

TABLE 3 Inhibition of RA-Induced Epidermal Hyperplasia with Compound H.Dosage of Percent Reduction in Retinoid- Compound H (μM) ToxicityInduced Hyperplasia (%). 0.5 None detected 40 + 10 1.0 None detected48 + 15 2.0 2.0 + 0.5 >95

Example 12 General Procedures

Human Epidermal Keratinocytes and Dermal Fibroblasts in MonolayerCulture.

Epidermal keratinocytes were isolated from fresh tissue biopsies asdescribed previously (Varani et al., (1994) J. Clin. Invest.94:1747-1753). Primary and early passage cells were maintained inKeratinocyte Growth Medium (KGM) (Lonza). KGM contains the same basalmedium as KBM but is further supplemented with a mixture of growthfactors including 0.1 ng per mL EGF, 0.5 μg per mL insulin, and 0.4%bovine pituitary extract. In addition to using low-passagekeratinocytes, we also used the HaCat line of immortalized humanepidermal keratinocytes in some experiments. The immortalizedkeratinocytes were handled exactly as low-passage keratinocytes.

Fibroblasts obtained from the same tissue were grown in monolayerculture using Dulbecco's modified minimal essential medium supplementedwith nonessential amino acids and 10% fetal bovine serum (DMEM-FBS) asculture medium. Both keratinocytes and fibroblasts were maintained at37° C. in an atmosphere of 95% air and 5% CO₂. Cells were subcultured byexposure to trypsin/ethylenediamine tetraacetic acid (EDTA) and used atpassage 2-4.

Proliferation Assays.

Keratinocyte proliferation was assessed by seeding 4×10⁴ cells per wellin a 24-well plate using KGM as culture medium. After the cells hadattached (overnight), they were washed and triplicate samples wereharvested for zero-time counts. The remaining cells were then incubatedin KGM with different concentrations of test reagents as indicated inthe Table 3. DMSO served as a negative control. Proliferation wasmeasured on day 2 by releasing the cells with trypsin/EDTA andenumerating them using a particle counter (Coulter Electronics, Hialeah,Fla.). Fibroblast proliferation studies were conducted in a similarmanner except that KBM supplemented with 1.4 mM Ca²⁺ was used as culturemedium.

Results:

The results of the cell proliferation assays are depicted in Table 4. KCrefers to human keratinocytes. HFF refers to human foreskin fibroblasts.The cLogP values were calculated using ChemDraw™ (CambridgeSoft).

TABLE 4

Percent Inhibition of Cells Per Com- Concentration of Test Compound (μM)pound R¹ R² R³ cLogP Cell 10 5 1 0.5 0.1 0.05 1 CH₃ 2,6- OH 5.57 KC ≧90≧90 ≧20 <20 <20 <20 dimethylbenzyl HFF ≧90 ≧90 <20 <20 <20 <20 2 CH₃3,5- OH 6.38 KC ≧90 ≧90 ≧20 ≧20 <20 <20 dimethylbenzyl HFF ≧90 ≧90 <20<20 <20 <20 3 H 2,6- OH 6.58 KC ≧90 ≧90 ≧20 <20 <20 <20 diethylbenzylHFF ≧90 ≧90 <20 <20 <20 <20 4 CH₃ 3,4- OH 6.48 KC ≧90 ≧90 ≧20 <20 <20<20 diethylbenzyl HFF ≧90 ≧90 <20 <20 <20 <20 5 H 3,5- OH 6.63 KC ≧90≧90 ≧20 ≧20 <20 <20 diethylbenzyl HFF ≧90 ≧90 <20 <20 <20 <20 6 CH₃ 3,5-OH 5.85 KC ≧90 ≧90 <20 <20 <20 <20 diethylbenzyl HFF ≧90 ≧90 <20 <20 <20<20 7 H 4- OH 6 KC ≧90 ≧90 ≧20 <20 <20 <20 isopropylbenzyl HFF ≧90 ≧90≧20 <20 <20 <20 8 CH₃ 4- OH 7.23 KC ≧90 ≧90 ≧20 ≧20 <20 <20isopropylbenzyl HFF ≧90 ≧90 <20 <20 <20 <20 9 H 2,5-diisopropyl OH 7.37KC ≧90 ≧90 ≧20 ≧20 <20 <20 benzyl HFF ≧90 ≧90 <20 <20 <20 <20 10 CH₃2,5-diisopropyl OH 5.46 KC ≧90 ≧90 ≧20 <20 <20 <20 benzyl HFF ≧90 ≧90<20 <20 <20 <20 11 H 3,4- F 7.11 KC ≧90 ≧90 <20 <20 <20 <20diethylbenzyl HFF ≧90 <20 <20 <20 <20 <20

Based on the data from the cell proliferation assays, compound H has anEC₅₀ of 0.7±0.1 M against proliferation of human keratinocyte cells andan EC₅₀ of 1.4±0.3 M against proliferation of human foreskinfibroblasts.

Example 13

The compounds described herein can be tested for activity againstF₁F₀-ATPase by measuring ATP synthesis and ATP hydrolysis. In addition,the compounds described herein can be evaluated for cytotoxicity againstRamos cells. Inhibition of ATP synthesis and hydrolysis by theF₁F₀-ATPase and cytotoxicity in Ramos cells can be measured as describedin K. M. Johnson et al. in Chemistry & Biology (2005) Vol. 12, pp.485-496. Testing of7-Chloro-5-(4-fluorophenyl)-3-(2-methylbenzyl)-1H-benzo[e][1,4]diazepin-2(3H)-oneusing these procedures indicated that this compound has an IC₅₀ of 9.1μM in inhibiting ATP synthesis by ATPase, and has an EC₅₀ of 7.9 μM incausing death of Ramos cells.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientificarticles referred to herein is incorporated by reference for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

I claim:
 1. A compound represented by formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹ is H or C₁₋₆alkyl; R² is ethyl; R³ is H or ethyl; R⁴ is hydroxyl or fluoro; whereinthe stereochemical configuration at a stereocenter in a compoundrepresented by formula I is R, S, or a mixture thereof.
 2. The compoundof claim 1, wherein R⁴ is fluoro.
 3. The compound of claim 1, having thestructure:


4. The compound of claim 1, wherein R⁴ is hydroxyl.
 5. The compound ofclaim 1, having the structure:


6. The compound of claim 1, having the structure:


7. The compound of any one of claims 1-6, wherein R² and R³ are ethyl.8. The compound of any one of claims 1-6, wherein R² is ethyl, and R³ isH.
 9. A pharmaceutical composition comprising a compound of claim 1, ora pharmaceutically acceptable salt thereof, a pharmaceuticallyacceptable carrier.
 10. The pharmaceutical composition of claim 9,further comprising a steroid, cyclosporine, vitamin D, vitamin D analog,keratolytic agent, topical retinoid, calcineurin inhibitor, or coal tar.11. The pharmaceutical composition of claim 10, wherein the steroid istriamcinolone acetonide or betamethasone dipropionate; the vitamin Danalog is calcipotriene; the keratolytic agent is anthralin; the topicalretinoid is tretinoin or tazarotene; and the calcineurin inhibitor istacrolimus, pimecrolimus, ascomycin, or ISA247.
 12. The pharmaceuticalcomposition of claim 9, further comprising a steroid.
 13. A method oftreating a skin condition selected from the group consisting of atopicdermatitis, rosacea, psoriasis, and a skin condition associated withepidermal hyperplasia, comprising administering a therapeuticallyeffective amount of a compound represented by formula I:

or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable carriers, wherein R¹ is H or C₁₋₆alkyl; R²is ethyl; R³ is H or ethyl; R⁴ is hydroxyl or fluoro; wherein thestereochemical configuration at a stereocenter in a compound representedby formula I is R, S, or a mixture thereof, to a subject in need thereofto ameliorate a symptom of the condition.
 14. The method of claim 13,wherein the skin condition is associated with epidermal hyperplasia. 15.The method of claim 13, wherein the skin condition is atopic dermatitis,rosacea or psoriasis.
 16. The method of claim 13, wherein the skincondition is psoriasis.
 17. The method of claim 13, wherein the compoundis a compound of claim 5 or
 6. 18. A method of treating epidermalhyperplasia, comprising administering a therapeutically effective amountof a compound of claim 1 to a subject in need thereof to ameliorate asymptom of the epidermal hyperplasia.
 19. The method of claim 18,wherein the compound is a compound of claim 5 or
 6. 20. The method ofclaim 13, wherein the subject is a human.
 21. The method of claim 13,further comprising administering to the subject a therapeutic agentselected from the group consisting of a steroid, cyclosporine, vitaminD, vitamin D analog, keratolytic agent, topical retinoid, calcineurininhibitor, and coal tar.
 22. The method of claim 21, wherein the steroidis triamcinolone acetonide or betamethasone dipropionate; the vitamin Danalog is calcipotriene; the keratolytic agent is anthralin; the topicalretinoid is tretinoin or tazarotene; and the calcineurin inhibitor istacrolimus, pimecrolimus, ascomycin, or ISA247.
 23. The method of claim21, wherein the therapeutic agent is a steroid.
 24. A method of reducingthe proliferation of a keratinocyte cell, comprising exposing said cellto a compound of claim
 1. 25. The method of claim 24, wherein thecompound is a compound of claim 5 or
 6. 26. The compound of claim 1,wherein the compound is selected from the group consisting of:(Z)-7-chloro-3-(2-ethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3-ethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(4-ethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,3-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,4-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,5-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,6-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,5-diethylbenzyl)-5-(4-hydroxyphenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2-ethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3-ethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(4-ethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,3-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,4-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,5-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,6-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,5-diethylbenzyl)-5-(4-hydroxyphenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2-ethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3-ethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(4-ethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,3-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,4-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,5-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,6-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,5-diethylbenzyl)-5-(4-fluorophenyl)-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2-ethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3-ethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(4-ethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,3-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,4-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,5-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(2,6-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;(Z)-7-chloro-3-(3,4-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one;and(Z)-7-chloro-3-(3,5-diethylbenzyl)-5-(4-fluorophenyl)-1-methyl-1H-benzo[e][1,4]diazepin-2(3H)-one.27. The compound of claim 1, wherein the compound is selected from thegroup consisting of:


28. The pharmaceutical composition of claim 9, wherein the one or morepharmaceutically acceptable carriers is selected from the groupconsisting of: phosphate buffered saline solution, water, oil/wateremulsion, water/oil emulsion, wetting agents, stabilizers, andpreservatives.
 29. The pharmaceutical composition of claim 28, whereinthe one or more pharmaceutically acceptable carriers is an oil/wateremulsion.
 30. A topical formulation comprising a compound represented byformula I:

or a pharmaceutically acceptable salt thereof, wherein R¹ is H orC₁₋₆alkyl; R² is C₁₋₆alkyl; R³ is H or C₁₋₆alkyl; R⁴ is hydroxyl orfluoro; wherein the stereochemical configuration at a stereocenter in acompound represented by formula I is R, S, or a mixture thereof, and andone or more pharmaceutically acceptable carriers.
 31. The topicalformulation of claim 30, wherein R⁴ is fluoro.
 32. The topicalformulation of claim 30, having the structure:


33. The topical formulation of claim 30, wherein R⁴ is hydroxyl.
 34. Thetopical formulation of claim 30, having the structure:


35. The topical formulation of claim 30, having the structure:


36. The topical formulation of claim 30, wherein R² and R³ are C₁₋₃alkyl.
 37. The topical formulation of claim 30, wherein R² and R³ aremethyl.
 38. The topical formulation of claim 30, wherein R² and R³ areethyl.
 39. The topical formulation of claim 30, wherein R² and R³ areisopropyl.
 40. The topical formulation of claim 30, wherein R² is H, andR³ is isopropyl.
 41. The topical formulation of claim 34, wherein R¹ isH or methyl; and R² and R³ are C₁₋₃ alkyl.
 42. The topical formulationof claim 30, wherein the topical formulation is an ointment, cream,suspension, lotion, powder, solution, paste, gel, spray, aerosol, oroil.
 43. The topical formulation of claim 42, wherein the topicalformulation is a cream.
 44. The topical formulation of claim 43, whereinthe topical formulation comprises at least 30% w/w of a polyhydricalcohol.